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Countess™ II FL automated cell counter
USER GUIDE
For fluorescence and brightfield applications
Catalog Number AMQAF1000
Document Part Number MP10826
Publication Number MAN0010644
Revision E.0
For Research Use Only. Not for use in diagnostic procedures.
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Life Technologies Corporation | 22025 20th Ave SE Ste. 100 | Bothell, WA 98021
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0010644
Revision Date Description
E.0 07 June 2019 Updated trypan blue handling instructions. Document converted to XML.
D.0 30 June 2017 Add info about edited profile indicator, save profile from results screen,
C.0 01 September 2015 Remove instructions for Countess II, update UI for the new SW version,
B.0 12 December 2014 Correct technical specification for cell size
A.0 08 September 2014 New user guide
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2019 Thermo Fisher Scientific Inc. All rights reserved.
dilution calculator, and reports.
rebrand
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Contents
■
CHAPTER 1 About this guide ............................................ 7
Audience ....................................................................... 7
User documentation ............................................................. 7
Text and keyboard conventions .................................................... 7
User attention words ............................................................ 8
Safety alert words ............................................................... 8
■
CHAPTER 2 Product information ....................................... 9
Product contents ................................................................ 9
Upon receiving the instrument ................................................ 9
Register your instrument .................................................... 9
Product description ............................................................ 10
Countess™ II FL automated cell counter ...................................... 10
Instrument exterior components ................................................. 11
■
CHAPTER 3 Getting started ............................................ 12
Installation .................................................................... 12
Operating environment ..................................................... 12
Install the instrument ...................................................... 12
Turn ON the instrument .................................................... 13
Load profile ................................................................... 14
Profiles screen ............................................................ 14
Automatic instrument functions ............................................. 14
Count parameters ......................................................... 15
Load a profile ............................................................. 16
Add/edit a profile .......................................................... 16
Display of profile names .................................................... 18
Prepare sample ................................................................ 19
Recommendations ......................................................... 19
Load Countess™ chamber slide .............................................. 19
Load Countess™ II FL reusable slide .......................................... 20
Slide operation ................................................................ 20
Countess™ cell counting chamber slide ....................................... 20
Countess™ II FL reusable slide .............................................. 21
Countess™ II FL automated cell counter User Guide
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Contents
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CHAPTER 4 Cell count and cell viability assays ...................... 23
Count cells in brightfield ........................................................ 23
Capture and count ......................................................... 23
Next steps ................................................................ 25
View results ................................................................... 25
Results screen for brightfield ............................................... 25
Identify objects counted ......................................................... 26
Advanced™ screen ......................................................... 26
Identify live and dead cells .................................................. 26
Graph count results ............................................................ 27
View graph ................................................................ 27
Gate count results .............................................................. 27
Adjust screen ............................................................. 27
Gate count results ......................................................... 27
Save as new protocol ........................................................... 29
Edit and save profile as new protocol ......................................... 29
■
CHAPTER 5 Fluorescence assays ..................................... 31
Count cell fluorescence ......................................................... 31
Overview ................................................................. 31
Count procedure ........................................................... 31
Next steps ................................................................ 33
View results ................................................................... 34
Results screen for cell fluorescence assays ................................... 34
Identify objects counted ......................................................... 34
Advanced™ screen ......................................................... 34
Identify cells counted in fluorescence assays .................................. 35
Graph count results ............................................................ 36
View graph for cell fluorescence assays ...................................... 36
Gate count results .............................................................. 37
Adjust screen ............................................................. 37
Gate count results ......................................................... 37
Save as new protocol ........................................................... 39
Edit and save profile as new protocol ......................................... 39
■
CHAPTER 6 Dilution calculator ........................................ 41
Calculate dilution .............................................................. 41
Dilution calculator ......................................................... 41
Calculate dilution .......................................................... 41
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Countess™ II FL automated cell counter User Guide
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■
CHAPTER 7 Save results ............................................... 44
Save count results ............................................................. 44
Save screen ............................................................... 44
Save procedure ............................................................ 44
Report ........................................................................ 47
Report file ................................................................ 47
■
CHAPTER 8 Instrument settings ...................................... 50
Overview ...................................................................... 50
Instrument settings screen ................................................. 50
Software update ............................................................... 50
Guidelines for software update .............................................. 50
Update the Countess™ II/II FL software ....................................... 51
Date/Time ..................................................................... 52
Set the date and time ....................................................... 52
Change light cube .............................................................. 54
Install or change EVOS™ light cube ........................................... 54
Contents
■
CHAPTER 9 Maintenance .............................................. 56
Instrument care ............................................................... 56
General guidelines for care ................................................. 56
Power supply .............................................................. 56
Clean the Countess™ II FL automated cell counter .................................. 56
Introduction ............................................................... 56
Clean the touch-screen ..................................................... 57
Clean the instrument case .................................................. 57
Decontaminate the instrument .............................................. 57
Set nominal focus .............................................................. 57
Overview ................................................................. 57
Set nominal focus .......................................................... 58
■
APPENDIX A Troubleshooting ......................................... 60
■
APPENDIX B Product specifications .................................. 63
Technical specifications ......................................................... 63
Physical characteristics .................................................... 63
Technical specifications .................................................... 63
Optics .................................................................... 63
Analysis slide ............................................................. 63
EVOS™ light cubes .............................................................. 64
LED illumination ........................................................... 64
EVOS™ light cubes ......................................................... 64
Countess™ II FL automated cell counter User Guide
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Contents
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APPENDIX C Ordering information .................................... 66
Countess™ II FL automated cell counter and accessories ............................ 66
Accessory products ............................................................ 66
■
APPENDIX D CSV file format ........................................... 68
CSV file format, explained ....................................................... 68
Overview ................................................................. 68
■
APPENDIX E Safety ..................................................... 71
Safety alert words .............................................................. 71
Electrical symbols ............................................................. 72
Safety symbols ................................................................ 72
Environmental symbols ......................................................... 73
Safety labels on instruments .................................................... 74
General instrument safety ...................................................... 74
Operating the instrument ................................................... 74
Safety precautions ......................................................... 75
Cleaning or decontaminating the instrument .................................. 75
Removing covers or parts of the instrument ................................... 75
Chemical safety ................................................................ 76
Chemical waste safety .......................................................... 76
Chemical waste hazard ..................................................... 76
Chemical waste safety guidelines ............................................ 77
Waste disposal ............................................................ 77
Electrical safety ................................................................ 77
Biological hazard safety ......................................................... 78
Safety and electromagnetic compatibility (EMC) standards .......................... 79
■
Documentation and support ............................................. 80
Customer and technical support ................................................. 80
Limited product warranty ....................................................... 80
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Countess™ II FL automated cell counter User Guide
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1
Audience
This user guide is for laboratory sta operating, maintaining, and analyzing data
using the Countess™ II FL Automated Cell Counter.
User documentation
The guides listed below are available for the Countess™ II FL Automated Cell
Counter.
About this guide
Countess™ II FL Automated Cell Counters User Guide
Countess™ II and Countess™ II FL Automated Cell Counters Quick
Reference Card (QRC)
Additional resources are available on the Countess™ Technical Resources page. Go to
www.thermosher.com/countess to access protocols, application notes, and tutorials.
Text and keyboard conventions
Text and keyboard conventions used in this user guide are listed below. For safety
alert words and symbols used in this document, see “Safety alert words“ on page 8.
Convention
Bold Bold text indicates user action. For example:
Press More.
► Right arrow symbol (►) indicates a menu choice, and separates
successive commands you execute or select from a drop-down or
shortcut menu. For example:
Guide
Pub. No.
MAN0010644
MAN0010826
Use
Select More ► Adjust.
Countess™ II FL automated cell counter User Guide
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Page 8
Chapter 1 About this guide
1
User attention words
User attention words
Two user aention words appear this document. Each word implies a particular level
of observation or action as described below.
Note: Provides information that may be of interest or help but is not critical to the use
of the product.
IMPORTANT! Provides information that is necessary for proper instrument
operation, accurate installation, or safe use of a chemical.
Safety alert words
Four safety alert words appear in this document at points where you need to be aware
of relevant hazards. Each alert word—IMPORTANT, CAUTION, WARNING,
DANGER—implies a particular level of observation or action, as dened below:
IMPORTANT! Provides information that is necessary for proper instrument
operation, accurate installation, or safe use of a chemical.
CAUTION! Indicates a potentially hazardous situation that, if not avoided, may
result in minor or moderate injury. It may also be used to alert against unsafe
practices.
WARNING! Indicates a potentially hazardous situation that, if not avoided,
could result in death or serious injury.
DANGER! Indicates an imminently hazardous situation that, if not avoided,
will result in death or serious injury. This signal word is to be limited to the
most extreme situations.
Except for IMPORTANT! safety alerts, each safety alert word in this document
appears with an open triangle gure that contains a hazard symbol. These hazard
symbols are identical to the hazard symbols that are axed to the instruments (see
Safety symbols in Appendix E).
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Countess™ II FL automated cell counter User Guide
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2
Product contents
Product information
The Countess™ II FL Automated Cell Counter is shipped with the following
components.
Upon receiving the instrument
Component
Countess™ II FL Automated Cell Counter (Cat. No. AMQAF1000) 1 each
Power Cord with 4 adaptor cords
(for U.S./Canada/Taiwan/Japan, Europe, or UK)
Countess™ Cell Counting Chamber Slides (50 slides/box) 1 box
Countess™ II FL Disposable Slide Holder 1 each
Countess™ II FL Reusable Slide Holder 1 each
Countess™ II FL Light Cube Removal Tool 1 each
Countess™ II USB drive 1 each
Countess™ II FL Automated Cell Counter Quick Reference Card 1 each
Examine the instrument carefully for damage incurred during transit. Ensure that all
parts of the instrument, including accessories listed above, are included with the
product. Damage claims must be led with the carrier; the warranty does not cover
in-transit damage.
See “Installation“ on page 12 for instructions on installing the instrument.
Quantity
1 each
Register your instrument
Countess™ II FL automated cell counter User Guide
Visit www.thermosher.com/registercountess to register your instrument. You will
be asked to supply the serial number, your name, and your contact details.
Registering your instrument ensures that you will receive notications of software
upgrades and information on new assays for use with the Countess™ II FL Automated
Cell Counter.
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Page 10
Chapter 2 Product information
2
Product description
Product description
Countess™ II FL automated cell counter
The Countess™ II FL Automated Cell Counter is a fully automated, 3-channel cell
counter and assay platform that uses EVOS™ light cube technology, state-of-the-art
optics, and image analysis algorithms to analyze uorescently labeled cells or trypan
blue stained samples in suspension.
• The Countess™ II FL Automated Cell Counter oers an intuitive user interface,
and provides the option to save data and generate a report, which can then be
transferred to a PC using the USB drive supplied with the instrument or available
separately.
• The cells to be counted are loaded into the instrument either in disposable
Countess™ Cell Counting Chamber Slides or in glass Countess™ II FL Reusable
Slides (“Prepare sample“ on page 19). Each chamber slide contains two enclosed
chambers to hold the sample to allow you to measure two dierent samples or
perform replicates of the same sample.
• The instrument takes 10 seconds per sample for a typical cell count in the
brighteld channel and is compatible with a wide variety of eukaryotic cells. In
addition to cell count and viability, the Countess™ II FL Automated Cell Counter
also provides information on cell size.
• In addition to the brighteld channel, the Countess™ II FL Automated Cell
Counter can accommodate two interchangeable EVOS™ uorescent light cubes
(“EVOS™ light cubes“ on page 64), enabling it to be used for multiple-
uorescence research applications.
• When equipped with EVOS™ light cubes, the Countess™ II FL Automated Cell
Counter can be used to perform uorescence assays for cells in suspension,
including simultaneous counts of cells stained with two dierent uorescent
dyes, GFP and RFP expression, apoptosis, and cell viability (live, dead, and total
cells). These assays are compatible with a wide variety of eukaryotic cells.
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Countess™ II FL automated cell counter User Guide
Page 11
Instrument exterior components
1
Touch-screen display: The 7‑inch capacitive touch-screen display is the main user
interface of the Countess™ II FL Automated Cell Counter. It contains the buttons for all
instrument functions and displays data from the cell count.
2
USB ports: The USB ports allow you to transfer and save the cell count data and image to
an external computer for record keeping and printing purposes. You can use the USB drive
supplied with the instrument or any other standard, FAT32-formatted USB drive for data
transfer. If desired, you can plug in a USB mouse into the rear USB port for instrument
control.
Note: The USB ports located in the front and the back of the instrument function the
same. However, the first USB drive connected will be the preferred saving location and
both USB drives cannot be accessed at the same time.
3
Slide port:
counter.
The Countess™ II FL instrument accepts both the disposable Countess™ Cell Counting
Chamber Slides and the glass Countess™ II FL Reusable Slides via interchangeable, slide-
specific carriers. For more information, see “Slide operation“ on page 20.
4
Back panel: The back panel of the Countess™ II FL Automated Cell Counter allows access
to the optional EVOS™ light cubes and provides storage for the light cube tool and the
reusable slide carrier. The back panel is secured to the instrument by two captive ¼-turn
fasteners.
5
Power switch: The ON/OFF rocker switch is the main power switch. It is not necessary to
use the power switch for day-to-day operation of the instrument.
6
EVOS™ light cubes: The EVOS™ light cubes allow the Countess™ II FL Automated Cell
Counter to analyze fluorescently labeled samples. The Countess™ II FL Automated Cell
Counter can accommodate two fluorescent light cubes. For more information, see “EVOS
light cubes“ on page 64.
7
USB ports: See 2 above.
8
Power input jack: The power input jack connects the instrument to an electrical outlet
through the supplied power cord and the appropriate plug, based on the electrical outlet
configuration in your country.
The slide port is used to insert the analysis slide containing the sample into the
Chapter 2 Product information
Instrument exterior components
2
™
Countess™ II FL automated cell counter User Guide
11
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3
Installation
Getting started
Operating environment
Install the instrument
• Place the instrument on a level surface away from vibrations emanating from
other pieces of equipment.
• Allow at least 5 cm (2 in) free space at the back of the instrument to allow for
proper ventilation and prevent overheating of electronic components.
• Set up the instrument away from direct light sources, such as windows. Ambient
room lighting can enter the imaging path and aect the image quality.
• Operating temperature range: 4°–32℃ (40°–90°F).
• Relative humidity range: <80%.
IMPORTANT! Do not position the instrument so that it is dicult to turn o the main
power switch located on the back of the instrument (“Instrument exterior
components“ on page 11).
In case of an instrument malfunction, turn the main power switch to the OFF position
and disconnect the instrument from the wall outlet.
Unpack the instrument and place the instrument on a at, level, dry surface.
1.
Remove the thin plastic protector lm from the touch-screen display.
2.
Plug one end of the power cord appropriate for your region into the instrument.
3.
Plug the power cord into the electrical outlet. Be sure to use only the power cord
4.
supplied with your instrument. Powering the instrument with an unapproved
power cord may damage the instrument.
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Countess™ II FL automated cell counter User Guide
Page 13
Turn ON the instrument
Chapter 3 Getting started
Turn on the instrument by ipping the power switch on the back of the
1.
instrument (“Instrument exterior components“ on page 11) to the ON position.
The instrument initializes and displays the Home screen.
Installation
3
From the Home screen, you can proceed immediately to the assays by inserting a
2.
slide (Chapter 4, “Cell count and cell viability assays“).
Alternatively, you can change or add a prole (Step 3 on page 13) or change
instrument seings (Step 4 on page 13).
To change the current prole or to add a new prole to the instrument, press the
3.
Proles
Proles allow you to create customized count preferences (i.e., gate counts based
on cell size, brightness, circularity, and/or relative uorescence intensity) (“Load
prole“ on page 14).
To change instruments seings, press the Instrument Seings buon in the
4.
upper right corner.
Instrument seings allow you to update the Countess™ II software, change the
date and time, and install or change up to two EVOS™ light cubes (Chapter 8,
“Instrument seings“).
buon in the upper left corner.
Countess™ II FL automated cell counter User Guide
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Chapter 3 Getting started
3
Load profile
Load profile
Profiles
screen
Proles screen allows you to create and save up to 9 customized proles. Each custom
prole denes the count parameters (size, brightness, circularity, and uorescence
intensity) and automatic instrument functions (Auto Lighting and Auto FL
Threshold) for a consistent and streamlined workow.
• You can access the Proles screen from the Home, Capture, Results, Advanced™,
or Adjust screens.
• The current prole is displayed on the upper left corner of the Home, Capture,
Results, Advanced™, or Adjust screens.
• Automatic instrument functions (below) and count parameters (“Count
parameters“ on page 15) are dened in the Edit prole screen (see “Add/edit a
prole“ on page 16).
• The Default prole contains default count seings and cannot be edited.
• The count parameters specied in the selected prole are applied to all new cell
counts.
• If you have already performed a count, loading a new prole from the Results
screen applies the count preferences to the current counts results (total cells,
viability etc.) and to all new counts.
• If you change any seing that is saved as part of the protocol (size, brightness, or
circularity) on the Results screen, the prole name is appended with the (*)
symbol.
Automatic instrument functions
14
You can turn the Auto FL Threshold and Auto Lighting functions ON and OFF using
the Auto FL Threshold and Auto Lighting checkboxes in the Edit prole screen
(“Add/edit a prole“ on page 16).
• Auto FL Threshold: Automatically applies threshold in uorescence channels to
subtract background uorescence for improved analysis despite variable
background levels between samples. This function is available only for
instruments equipped with the optional EVOS™ light cubes.
• Auto Lighting: Automatically illuminates the sample in the brighteld for
increased sample-to-sample consistency and decreased user-to-user variability.
Countess™ II FL automated cell counter User Guide
Page 15
Chapter 3 Getting started
Load profile
3
Count parameters
Count parameters are adjusted in the Edit prole screen using the parameter sliders.
Parameter sliders correspond to a single channel, which is selected using the channel
selection radio buons located above the sliders.
• Size: As you move the slider up, the algorithm includes larger objects in the
count. As you move the slider down, only the smaller objects are counted.
• Brightness: As you move the slider up, the algorithm includes the brighter
objects in the count. As you move the slider down, only the dimmest of objects
are counted.
• Circularity: As you move the slider up, the algorithm includes more objects with
shapes other than circular in the count. As you move the slider down, only the
objects that are perfect circles are counted.
• Fluorescence intensity: As you move the slider up, the algorithm includes the
objects that uoresce more brightly in the count. As you move the slider down,
only the dimmest of objects are counted.
• Size, brightness, and uorescence intensity sliders are range sliders.
To adjust the upper and lower boundaries without changing the data range, drag
the slider by its middle section (i.e., the slider bar).
To adjust only the upper or the lower boundary, move the upper or the lower
handle in the desired direction. This will also change the range of values within
which the cells are counted.
• The circularity slider only sets a single threshold value; cells that fall below the
set value are counted, and cells that are beyond this range are excluded.
To adjust the threshold for circularity, drag the slider in the desired direction.
Countess™ II FL automated cell counter User Guide
15
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Chapter 3 Getting started
3
Load profile
Load a profile
Add/edit a profile
Press the Proles buon located on the upper left corner of the screen to open
1.
the Proles screen.
Press the desired prole to select, and then press Load.
2.
The instrument will load the count parameters specied in the selected prole
and return to the previous screen.
To return to the previous screen without loading the new prole, press the
3.
previous
The instrument will keep the saved prole, but return to the previous screen
without loading it.
Press the Proles buon located on the upper left corner of the screen to go to
1.
the Proles screen.
To add or edit a new prole, select an empty or an existing prole, and then
2.
press Edit. The Edit screen for the selected prole opens.
Note: The Default prole contains default count seings and cannot be edited.
Select or deselect the Auto FL Threshold checkbox to turn the Auto FL
3.
Threshold function ON or OFF (“Automatic instrument functions“ on page 14).
buon.
Note: This function is available only for instruments equipped with the optional
EVOS™ light cubes.
Select or deselect the Auto Lighting checkbox to turn the Auto Lighting function
4.
ON or OFF (“Automatic instrument functions“ on page 14).
Define count parameters for the brightfield channel:
To dene the new count parameters in the brighteld channel:
From the Trypan Blue selection box, select the Live or Dead radio buon.
1.
Adjust the size, brightness, and circularity thresholds using the corresponding
2.
parameter slider (“Count parameters“ on page 15).
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Countess™ II FL automated cell counter User Guide
Page 17
Chapter 3 Getting started
Load profile
Define count parameters for the fluorescent channels
To dene the new count parameters for uorescence assays (available only for
1.
instruments equipped with the optional EVOS™ light cubes):
From the FL selection box, select the desired channel using the
a.
corresponding radio buon.
The available options are BF (brighteld) and up to two uorescence
channels, depending on the light cubes installed (GFP and TxRed in the
following example).
Dene the new count parameters for the selected channel using the
b.
corresponding parameter slider (“Count parameters“ on page 15).
Parameter sliders in the BF channel allow you to gate count results based on
size, brightness, and circularity.
The parameter slider in the selected uorescence channel allows you to gate
count results based on relative uorescence intensity in that channel.
Repeat for the remaining channels, as necessary.
c.
3
Note: You can further adjust the size, brightness, or circularity parameters
for the selected prole before or after performing a cell count, as needed.
You can then save these additional changes to the count parameters to the
current prole or as a separate prole directly from the Advanced™ screen
(see “Save as new protocol“ on page 29)
Countess™ II FL automated cell counter User Guide
17
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Chapter 3 Getting started
3
Load profile
To assign a name to the new prole or to change the name of the existing prole,
2.
press the Prole name text box. The alpha-numeric keypad opens.
Type in the desired prole name using the alpha-numeric keypad.
3.
To enter symbols, press the symbol (@%&) key. To return to the alpha-numeric
keypad, press ABC.
Press Enter to save the name and return to the Edit prole screen.
4.
To return to the Edit prole screen without saving the name, press the close
buon.
Display of
names
profile
Press Save to save the new prole, and then press Close in the conrmation
5.
screen to return to the Proles screen.
To return to the Proles screen without saving, press Cancel.
On the Proles screen, press Load. The instrument will load the count
6.
parameters specied in the selected prole and return to the previous screen.
To return to the previous screen without loading the new prole, press the
7.
previous
previous screen without loading it.
• The maximum display length for prole names on the screen is seven characters.
• If the prole name is over seven display characters, the name is shortened to the
rst seven characters with “…” at the end. For example, “MyProle” is shortened
to “MyPro…”.
• If you change any seing that is saved as part of the protocol (size, brightness, or
circularity) on the Results screen, the prole name is appended with the “(*)”
symbol.
For example, “Count” becomes “Count (*)”.
• When a prole with name of over four or ve display characters is edited, only
the rst four or ve characters is displayed and the name is appended with “…
(*)”.
For example, “MyProle” becomes “MyPro…(*)”.
buon. The instrument will keep the saved prole, but return to the
18
Countess™ II FL automated cell counter User Guide
Page 19
Prepare sample
Chapter 3 Getting started
Prepare sample
3
Recommendations
Load Countess
™
chamber slide
To obtain the best results, follow these recommendations:
• Ensure that the cell sample is homogeneously mixed.
• The measurement range extends from 1 × 104–1 × 107 cells/mL, but the optimal
range is 1 × 105–4 × 106 cells/mL.
• For accurate results in cell viability assays, ensure that the counting area is
covered with the cell suspension and count the cells immediately after staining
per the assay protocol.
• Do not press the optical surfaces of the chamber slides. Hold the slides by the
edges.
• Take care to avoid forming bubbles in the sample.
• Sterile ltering and centrifugation can be used to remove precipitates common
within trypan blue solutions. Alternatively, avoid mixing and vortexing trypan
blue stock solutions to allow precipitates to remain at the boom of tube, thereby
promoting more accurate cell counts. Also, precipitates can be reduced by gentle
heating at 37°C for 10 minutes.
Prepare the sample by adding 10 µL of your cell suspension to 10 µL of 0.4%
1.
trypan blue stain. Mix the sample mixture well by pipeing it up and down a
few times.
Gently pipet 10 µL of the sample into the half moon-shaped sample loading area.
2.
The sample is loaded into the chamber through capillary action.
Let the sample mixture sele in the chamber for 30 seconds, and then insert the
3.
slide into the slide port (“Instrument exterior components“ on page 11). You will
hear a soft click, if the slide is pushed in correctly.
To remove the slide, push the slide gently into the instrument until it “clicks”
4.
and a spring pushes the slide out. Grasp the slide and pull it out the rest of the
way.
Note: After using the Countess™ Cell Counting Chamber Slides, appropriately
dispose of them as biohazardous waste. Do not reuse the disposable chamber
slides.
Countess™ II FL automated cell counter User Guide
19
Page 20
Chapter 3 Getting started
3
Slide operation
Load Countess™ II FL reusable slide
Before loading your sample into the Countess™ II FL Reusable Slide, place a
1.
cover slip on the counting chamber, making sure the cover slip is clean and free
of grease.
Gently pipet 10 µL of the sample into the sample inlet, allowing capillary action
2.
to draw the sample into the counting chamber. A properly loaded counting
chamber should have a thin, even lm of uid under the cover slip.
After using the Countess™ II FL Reusable Slide, rinse the glass slide and cover
3.
slip with water, and then clean with 70% ethanol. Use Kimwipes™ laboratory
tissues to clean and dry the slides, as needed.
Slide operation
Countess™ cell counting chamber slide
Note: Each chamber in the Countess™ Cell Counting Chamber Slide or the
Countess™ II FL Reusable Slide has a 10-µL sample capacity. Do not overll the
slide chambers.
The Countess™ II FL instrument accepts both disposable Countess™ Cell Counting
Chamber Slides and glass Countess™ II FL Reusable Slides on interchangeable, slide-
specic carriers.
To use the plastic, disposable Countess™ Cell Counting Chamber Slide with the
1.
Countess™ II FL Automated Cell Counter, insert the slide carrier (black, see
image) into the slide port of the instrument until it clicks into place.
Note: The Countess™ II FL Automated Cell Counter is shipped with the
disposable slide carrier already installed
20
Countess™ II FL automated cell counter User Guide
Page 21
Chapter 3 Getting started
Slide operation
3
Countess™ II FL reusable slide
Load the chamber slide with your sample as described in “Load Countess
2.
chamber slide“ on page 19, and then insert the slide into the slide carrier in the
slide port until it clicks into place.
To remove the slide, push the slide gently into the instrument until it “clicks”
3.
and a spring pushes the slide out. Grasp the slide and pull it out the rest of the
way.
Optional: To remove the slide carrier, gently squeeze the tabs and pull the carrier
4.
completely out of the instrument.
Note: You can store the slide carrier behind the access panel on the back of the
instrument (“Instrument exterior components“ on page 11).
To use the Countess™ II FL Reusable Slide, unlatch the back panel of the
1.
Countess™ II FL Automated Cell Counter with the two captive ¼-turn fasteners
that secure the back panel on the rear of the instrument.
™
Remove the reusable slide carrier (white) from inside of the back panel.
2.
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21
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Chapter 3 Getting started
3
Slide operation
Load the reusable glass slide with the sample as described in “Load Countess™ II
3.
FL reusable slide“ on page 20, and place the loaded slide into the white slide
carrier.
Insert the carrier and reusable slide assembly into the slide port, and gently push
4.
into the instrument until it clicks into place.
To remove the slide, push the slide gently into the instrument until it clicks and a
5.
spring pushes the slide out. Grasp the slide and pull it out the rest of the way.
Optional: To count the second sample present on the reusable slide, simply
6.
remove the slide from the carrier, rotate, and reinsert the slide into the carrier so
that the second sample is aligned with the sample viewing hole.
Note: You can store the slide carrier behind the access panel on the back of the
instrument (“Instrument exterior components“ on page 11).
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Cell count and cell viability assays
4
Count cells in brightfield
Prepare the sample by adding 10 µL of your cell suspension to 10 µL of 0.4%
Capture and count
1.
trypan blue stain. Mix the sample mixture well by pipeing up and down a few
times.
Load 10 µL of the sample mixture per chamber into the sample slide as described
2.
in “Load Countess™ chamber slide“ on page 19. Let the sample mixture sele for
30 seconds.
Optional: Press the Proles
3.
described in “Load a prole“ on page 16.
Insert the sample slide into the slide port (“Instrument exterior components“ on
4.
page 11), making sure that the sample side is inserted completely into the
instrument. You will hear a soft click, if the slide is pushed in correctly.
When the slide is inserted, the instrument automatically illuminates the sample,
5.
sets the intensity of brighteld illumination, and auto focuses on the cells.
Note: To turn o the Auto Lighting function, see “Dene count parameters for
the uorescent channels“ on page 17.
buon and load the desired prole as
Optional: To manually adjust the focus, press the Focus™ buon, and then use
6.
the Focus™ slider to bring your sample into focus as described in “Set nominal
focus“ on page 57.
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Chapter 4 Cell count and cell viability assays
4
Count cells in brightfield
Press the Set buon to set the focus and collapse the focus controls.
7.
Once the focus has been set, the Set buon on the focus slider becomes inactive,
conrming that the focus seing has been stored.
Note: If needed, Zoom in on the image to adjust focus or lighting.
Optional: Set exposure using the light source slider.
8.
The light source slider controls the LED intensity, camera gain, and exposure
time and allows you to adjust the image brightness.
Note: If your instrument is equipped with an EVOS™ light cube, rst press
Adjust, and then select brighteld (white circle) as the light source. Set the
exposure, then press Done to return to the Capture screen.
Press Capture.
9.
Note: If your instrument is equipped with an EVOS™ light cube, make sure that
only the BF (brighteld) checkbox is selected under Collect channels before
capturing the image.
The instrument temporarily captures the image and displays the results (total
concentration, percentage and concentration of live and dead cells). For more
information, see “View results“ on page 25.
24
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Chapter 4 Cell count and cell viability assays
View results
4
Next steps
View results
Results screen for
brightfield
• To identify the objects (i.e., cells) counted as Live or Dead, press More to go to the
Advanced™ screen (“Identify objects counted“ on page 26).
• To see the distribution of live and dead cells in a graphical format, press the
Graph buon (“Graph count results“ on page 27).
• To gate the results by object size, brightness, or circularity, rst press More to
open the Advanced™ screen, then press Adjust to go to the Adjust screen (“Gate
count results“ on page 27).
Note: You can save the changes you make to the size, brightness, or circularity
parameters in the Adjust screen to the current prole or as a separate prole
directly from the Advanced™ screen (see “Save as new protocol“ on page 29).
• To calculate the volume of cell sample and buer needed to reach a desired
concentration based on the count results, press Dilution Calculator to open the
Dilution Calculator application (Chapter 6, “Dilution calculator“)
• To permanently save the results, press Save (Chapter 7, “Save results“).
• To perform a new count, remove the slide and reinsert it second chamber rst
into the instrument, or insert a new sample slide.
The Results screen for cell count and cell viability assays performed using the
brighteld channel displays a composite image of the objects counted and the results
of the cell count and cell viability calculations (total concentration, percentage and
concentration of live and dead cells).
Note: When performing cell counts and cell viability assays in brighteld, the
counting algorithm assumes that you have diluted your cells 1:1 in trypan blue and
takes this dilution into account when calculating the total cell concentration. The cell
concentration displayed in the Results screen is the original cell concentration before
dilution into trypan blue.
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Chapter 4 Cell count and cell viability assays
4
Identify objects counted
Identify objects counted
Advanced
™
screen
Identify live and dead cells
The Advanced™ screen allows you to identify the objects (i.e., cells) counted in each
channel and included in the count results for further review. After reviewing the
marked objects, you can adjust the threshold for size, brightness, and/or circularity as
desired for your application (“Gate count results“ on page 27).
On the Results screen, click More. The Advanced™ screen opens.
1.
To identify the cells that are included in the count as live, press the Live toggle
2.
buon. Live cells will be circled in green on the screen.
To identify the cells that are included in the count as dead, press the Dead toggle
buon. Dead cells will be circled in red on the screen.
Note: You may select either or both options. In the following example, both Live
and Dead buons are pressed and live and dead cells are marked with green and
red circles, respectively.
To unmark the cells identied as live (green) or dead (red) on the screen, press
3.
the Live or the Dead toggle buon again, respectively.
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Graph count results
Chapter 4 Cell count and cell viability assays
Graph count results
4
View graph
For cell count and cell viability assays performed in the brighteld channel, you can
view the distribution of cells (live and/or dead) based on size in a graphical format.
Note: You can view the Graph on Results, Advanced™, and Adjust screens.
To view the graph showing the distribution live and/or dead cells based on cell
1.
size, press the Graph buon.
To view the distribution of only the live or dead cells, check the corresponding
2.
Live or Dead check box on the graph.
The graph will automatically update and display the distribution of cells based
on size only in the selected population.
Optional: Using the size, brightness, and circularity sliders, adjust the count
3.
parameters. As you adjust the count parameters, the count results and the graph
will be automatically updated.
To close the graph, press the Graph buon again.
4.
Gate count results
Adjust screen
Gate count results
Countess™ II FL automated cell counter User Guide
The Adjust screen for cell count and cell viability assays in the brighteld channel
contains the controls for gating results based on size, brightness, and circularity. You
can adjust the count parameters before or after performing a count, and save these
changes to the current prole or as a separate prole (“Save as new protocol“ on
page 29).
On the Results screen, press More to open the Advanced™ screen.
1.
Optional: Press the Live and/or the Dead buon to identify the cells in the
2.
selected population (“Identify objects counted“ on page 26).
27
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Chapter 4 Cell count and cell viability assays
4
Gate count results
On the Advanced™ screen, press Adjust to open the Adjust screen.
3.
Optional: Press the Graph buon to view the distribution of cells (live and/or
4.
dead) based on size as you gate the count results (“Save as new protocol“ on
page 29).
Select the channel (Live or Dead) you wish to gate.
5.
Using the size, brightness, and circularity sliders, adjust the count parameters.
6.
Note: For a description of the count parameters and count parameter controls
(i.e., parameter sliders), see “Count parameters“ on page 15.
When nished, press Done to save the changes to count parameters and return
7.
to the Advanced™ screen.
Press Cancel to return to the Results screen without saving the changes.
On the Advanced™ screen, press Count to recalculate your results with the new
8.
count parameters.
To save the changes to size, brightness, or circularity parameters to the current
9.
prole or to create a prole with the new count parameters, see “Save as new
protocol“ on page 29.
To permanently save your results, see Chapter 7, “Save results“.
10.
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Countess™ II FL automated cell counter User Guide
Page 29
Save as new protocol
If you have made any changes to the count parameters before or after performing
Edit and save
profile as new
protocol
1.
a count, the displayed prole name is appended with the (*) symbol and the
Advanced™ results screen displays the New protocol buon, which allows you
to save the changes to the current prole or as a separate protocol.
Chapter 4 Cell count and cell viability assays
Save as new protocol
4
To save the changes to the count parameters to the current prole or to create a
2.
new prole with the edited parameters, press the New protocol buon. The
Select prole to edit screen opens.
Note: By default, the current prole buon is selected on the Select prole
screen. If you are using the Default prole for the count, no prole buon is
selected on this screen.
Select the prole you wish to edit, then press Import seings.
3.
Note: You can select only a previously saved or an empty prole. The Default
prole cannot be edited.
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Chapter 4 Cell count and cell viability assays
4
Save as new protocol
The Edit prole screen opens and displays the edited count parameters from the
4.
Adjust screen (“Gate count results“ on page 27).
Note: If you have selected a prole that had been previously saved, the name of
that prole populates the Prole name text box by default. Otherwise, the
textbox remains empty.
To change the name of the selected prole, press the Prole name text box and
5.
enter the desired name using the alpha-numeric keypad as described in
“Display of prole names“ on page 18.
Optional: If desired, make additional changes to the prole and the count
6.
parameters as described in “Add/edit a prole“ on page 16.
Click Save to save the new prole seings and return to the Results page for the
7.
last count. The prole name will be displayed without the (*) symbols.
Click Cancel to return to the Results page for the last count without saving the
changes to the prole. The prole name will be displayed with the (*) symbol,
indicating that the count parameters for the selected prole had been altered, but
not yet saved.
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5
Count cell fluorescence
Fluorescence assays
Overview
Count procedure
Countess™ II FL Automated Cell Counter equipped with the optional EVOS™ light
cubes can be used for a variety of uorescent applications, including simultaneous
counts of cells stained with two dierent uorescent dyes, GFP and RFP expression,
and apoptosis and cell viability assays.
For instructions on installing EVOS™ light cubes to your Countess™ II FL Cell
Counter, see “Change light cube“ on page 54.
Ensure that your uorescent cell sample is homogeneously mixed.
1.
Load 10 µL of the uorescent sample mixture per chamber into the sample slide
2.
as described in “Load Countess™ chamber slide“ on page 19. Let the sample
mixture sele for 30 seconds.
Optional: Press the Proles buon located on the upper left corner of the
3.
screen to open the Proles screen and load the desired prole as described in
“Load prole“ on page 14.
Insert the sample slide into the slide port (“Instrument exterior components“ on
4.
page 11), making sure that the sample side is inserted completely into the
instrument. You will hear a soft click, if the slide is pushed in correctly.
When the slide is inserted, the instrument automatically illuminates the sample,
5.
sets the intensity of brighteld illumination, and auto focuses on the cells.
Note: To turn o the Auto Lighting function, see “Dene count parameters for
the uorescent channels“ on page 17.
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31
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Chapter 5 Fluorescence assays
5
Count cell fluorescence
Optional: To manually adjust the focus, press the Focus™ buon and use the
6.
Focus™ slider to bring your sample into focus as described in “Set nominal
focus“ on page 57.
Press the Set buon to set the focus and collapse the focus controls. Once the
7.
focus has been set, the Set buon on the focus slider becomes inactive,
conrming that the focus seing has been stored.
To view your sample under a dierent light source, press the desired Light
8.
source buon. The instrument displays the sample in the selected channel
(brighteld or uorescent).
In the example below, the sample is displayed in the GFP channel.
Note: The light source buons select the light channel (brighteld and/or
uorescence) for sample illumination and are used when seing the exposure for
the selected channel (Steps 9 on page 32–11 on page 33); they do not determine
which channels are used for capturing the image.
To set exposure, press Adjust to go to the Adjust screen.
9.
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Chapter 5 Fluorescence assays
Press the light source buon for the channel you wish to set exposure and adjust
10.
the exposure using the light source slider. Repeat the procedure for the
remaining channels, if desired.
After seing the exposure, press Done to return to the Capture screen.
11.
To return to the Capture screen without changing the exposure, press Cancel.
On the Capture screen, select the Collect channels check boxes for the channels
12.
you wish to capture.
Press Capture.
13.
The instrument temporarily captures the image and displays the results (total
concentration, percentage and concentration of cells counted in each uorescence
channel). For more information, see “View results“ on page 34.
Count cell fluorescence
5
Next steps
Countess™ II FL automated cell counter User Guide
• To identify the objects (i.e., cells) counted in each channel, press More to go to the
Advanced™ screen (“Identify objects counted“ on page 34).
• To see the distribution of cells counted through each channel in a graphical
format, press the Graph buon (“Graph count results“ on page 36).
• To gate the results by object size, brightness, circularity, or relative uorescence
intensity, rst press More to open the Advanced™ screen, then press Adjust to go
to the Adjust screen (“Gate count results“ on page 37).
Note: You can save the changes you make to the size, brightness, circularity, and
relative uorescence intensity parameters in the Adjust screen to the current
prole or as a separate prole directly from the Advanced™ screen (see “Save as
new protocol“ on page 39).
• To calculate the volume of cell sample and buer needed to reach a desired
concentration based on the count results, press Dilution Calculator to open the
Dilution Calculator application (Chapter 6, “Dilution calculator“)
33
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Chapter 5 Fluorescence assays
5
View results
View results
• To permanently save the results, press Save (Chapter 7, “Save results“).
• To perform a new count, remove the slide and reinsert it second chamber rst
into the instrument, or insert a new sample slide.
Results screen for
cell fluorescence
assays
The Results screen for cell uorescence assays displays a composite image of the
objects counted and the results of the cell count and cell viability calculations (total
concentration, percentage and concentration of cells counted through each
uorescence channel).
Note: The total cell concentration displayed after a uorescent count does not take
any dilution into account. Therefore, the results reect the actual cell concentration in
the sample slide, which must be multiplied by any dilution factor present to calculate
the original cell concentration.
This is in contrast to the cell counts in brighteld, where the counting algorithm
assumes a 1:1 dilution of the sample in trypan blue and displays the original cell
concentration (i.e., before the dilution) in the Results screen.
Identify objects counted
Advanced™ screen
34
The Advanced™ screen allows you to identify the objects (i.e., cells) counted in each
channel and included in the count results for further review. After reviewing the
marked objects, you can adjust the threshold for size, brightness, and/or circularity as
desired for your application.
Countess™ II FL automated cell counter User Guide
Page 35
Identify cells
counted in
fluorescence
assays
Chapter 5 Fluorescence assays
On the Results screen, click More to open the Advanced™ screen.
1.
Optional: To view your sample under a specic light source (brighteld and/or
2.
uorescent), select the desired Channels checkbox (brighteld in the example
above). You may display your sample in any or all of the available channels.
Identify objects counted
5
To identify the cells that are counted in a specic channel, press the
3.
corresponding boundaries buon. Cells counted in the selected channel will be
circled on the screen with the same color as the selected channel.
In the example below, both the GFP and TxRed boundaries buons are pressed
and the cells counted in the GFP and TxRed channels are marked with green and
red circles, respectively.
To unmark the cells counted in a specic channel, press the corresponding
4.
boundaries buon again.
Countess™ II FL automated cell counter User Guide
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Chapter 5 Fluorescence assays
5
Graph count results
Graph count results
View graph for cell
fluorescence
assays
For uorescence assays, you have the option of viewing the distribution of the cells
based on size or based on relative uorescence intensity in a graphical format.
Note: You can view the Graph on Results, Advanced™, and Adjust screens.
To view the graph showing the distribution of cells based on size, press the
1.
Graph buon, and then select BF (brighteld).
The graph displays the size distribution of the total cell count (number of cells vs.
cell size in µm), and the average size of the cells counted in each available
uorescence channel.
36
To view the distribution of cells based on relative uorescence intensity, press the
2.
Graph buon, and then select FL (uorescence).
The graph displays the distribution of cells based on uorescence intensity.
Countess™ II FL automated cell counter User Guide
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Chapter 5 Fluorescence assays
To remove the cells counted in a specic channel from the graph, uncheck the
3.
corresponding channel check box on the graph.
The graph automatically updates and displays the distribution of cells based on
relative uorescence intensity only in the selected (i.e., checked) channel.
Gate count results
5
4.
5.
Gate count results
Adjust screen
Gate count results
The Adjust screen for cell uorescence assays contains the controls for gating count
results based on size, brightness, circularity, and uorescence intensity. You can adjust
the count parameters before or after performing a count, and save these changes to
the current prole or as a separate prole (“Save as new protocol“ on page 39).
1.
2.
3.
To add the cells counted in a specic channel to the graph, re-check the
corresponding channel check box.
To close the graph, press the Graph buon again.
On the Results screen, press More to open the Advanced™ screen.
Optional: To view your sample under a specic light source (brighteld and/or
uorescent), select the desired Channels checkbox on the Advanced™ screen
(“Identify cells counted in uorescence assays“ on page 35).
Optional: Press the desired boundaries buons on the Advanced™ screen to
identify the cells counted in the corresponding channel (“Identify cells counted
in uorescence assays“ on page 35).
Countess™ II FL automated cell counter User Guide
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Chapter 5 Fluorescence assays
5
Gate count results
Press Adjust to open the Adjust count parameters screen, which contains the
4.
controls for adjusting the count parameters in the selected channel.
Note: For a description of the count parameters and count parameter controls
(i.e., parameter sliders), see “Count parameters“ on page 15.
Optional: Press the Graph buon to view the distribution of cells based on
5.
size or uorescence intensity (“Graph count results“ on page 36).
Select the brighteld channel (white circle) to adjust the thresholds for size,
6.
brightness, and circularity using the size, brightness, and circularity sliders.
Select the desired uorescence channel (colored circles) to adjust the threshold
7.
for uorescence intensity using the uorescence intensity slider.
Note: The uorescence channels available depend on the EVOS™ light cubes
installed in the instrument.
When nished, press Done to save the changes to count parameters and return
8.
to the Advanced™ screen.
Press Cancel to return to the Results screen without saving the changes.
On the Advanced™ screen, press Count to recalculate your results with the new
9.
count parameters.
To save the changes to size, brightness, or circularity parameters to the current
10.
prole or to create a prole with the new count parameters, see “Save as new
protocol“ on page 39.
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Page 39
Save as new protocol
If you have made any changes to the count parameters, the displayed prole
Edit and save
profile as new
protocol
1.
name is appended with the (*) symbol and the Advanced™ results screen
displays the New protocol buon, which allows you to save the changes to the
current prole or as a separate protocol.
Chapter 5 Fluorescence assays
Save as new protocol
5
To save the changes to the count parameters to the current prole or to create a
2.
new prole with the edited parameters, press the New protocol buon. The
Select prole to edit screen opens.
Note: By default, the current prole buon is selected on the Select prole
screen. If you are using the Default prole for the count, no prole buon is
selected on this screen, because the Default prole cannot be edited.
Select the prole you wish to edit, then press Import seings.
3.
Note: You can select only a previously saved or an empty prole. The Default
prole cannot be edited.
Countess™ II FL automated cell counter User Guide
39
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Chapter 5 Fluorescence assays
5
Save as new protocol
The Edit prole screen opens and displays the edited count parameters from the
4.
Adjust screen (“Gate count results“ on page 37).
Note: If you have selected a prole that had been previously saved, the name of
that prole populates the Prole name text box by default. Otherwise, the
textbox remains empty.
To change the name of the selected prole, press the Prole name text box and
5.
enter the desired name using the alpha-numeric keypad as described in
“Display of prole names“ on page 18.
Optional: If desired, make additional changes to the prole and the count
6.
parameters as described in “Dene count parameters for the uorescent
channels“ on page 17.
Click Save to save the new prole seings and return to the Results page for the
7.
last count. The prole name will be displayed without the (*) symbols.
Click Cancel to return to the Results page for the last count without saving the
changes to the prole. The prole name will be displayed with the (*) symbol,
indicating that the count parameters for the selected prole had been altered, but
not yet saved.
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6
Calculate dilution
Dilution calculator
Dilution calculator
Calculate dilution
Dilution calculator function allows you to calculate the volume of cell sample and
buer needed to reach a desired concentration using the count results.
On the Results screen, press Dilution Calculator to open the Dilution calculator
1.
screen.
Press the cell type buon located to the right of the current concentration, then
2.
select the count result you wish to use for the dilution calculation from the
dropdown. The current concentration changes to reect the results for the cell
type selected.
Available Cell type options are:
• For cell count and viability assays in brighteld:
Live cells or Total cells.
By default, Live cells is selected.
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Chapter 6
6
Calculate dilution
Dilution calculator
Note: Channel 1 and Channel 2 name displayed in the dropdown menu
depends on the EVOS™ light cube installed. In the following example, GFP and
TxRed cubes have been installed and the dropdown displays GFP and TxR.
Enter the desired cell concentration (What is your desired cell concentration?):
3.
• For uorescence assays: Channel 1, Channel 2, or Total cells (Channel 1 +
Channel 2).
By default, Total cells is selected.
Press the value text box, then enter the value using the number pad.
a.
You can enter a one digit to the left of the decimal separator (integer part)
and one to the right (fractional part). If you do not enter the fractional part,
the software enters a 0 by default.
Press Enter or touch anywhere outside the number pad to close it.
Press the exponent buon, then select the exponent value for the desired
b.
concentration.
By default, the exponent is (n–1), where n = the exponent value of the
current count. The maximum selectable exponent is same exponent value as
the current count.
Pressing an exponent buon selects that exponent and closes the window.
Pressing anywhere outside the window keeps the previously selected
exponent and closes the window
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Chapter 6
Press the total volume text box (How many mL do you need?), then enter the
4.
total volume of the sample you wish to have at the new concentration using the
number pad.
By default, the total volume box is blank. The maximum volume you can enter is
999.9 mL and you can use only a single decimal.
Press Enter or touch anywhere outside the number pad to close the number pad.
When you have made valid entries for cell type, desired cell concentration, and
5.
total volume, and closed the last popup window, the boom line of the dilution
calculator displays the volumes of cell solution and buer needed.
Dilution calculator
Calculate dilution
6
If you enter a combination of values that is not valid (e.g., desired concentration
is greater than the current concentration), the results line remains on (or return
to) the blank state, and screen displays a warning message.
If you make any changes to any of the input areas above, the results are
recalculated automatically upon closing the popup window.
Press Done or the Back buon to return to the main Results screen.
6.
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7
Save count results
Save results
Save screen
Save procedure
The Countess™ II FL Automated Cell Counter allows you to save your data and
images using a USB ash drive.
To save your experiment, choose from the following options, in any combination:
• Result: Saves the Results screen as it is displayed on the instrument, with or
without the Graph, in the selected image format (JPEG, BMP, PNG, or TIFF).
• Images: Saves only the raw captured image in the selected image format (JPEG,
BMP, PNG, or TIFF).
• Data: Saves the data from the experiment as a CSV le (comma separated values).
The CSV format allows for processing or re-displaying results with any third
party software or spreadsheet program. For more information on the CSV le
format, see Appendix D, “CSV le format“.
• Report: Saves a printer-friendly report of the results, graph(s), and image in the
selected format (PDF, PNG, or JPEG). For more information, see “Report“ on
page 47.
Note: If you wish to save your results with the Graph showing the distribution
of cells based on cell size or uorescence intensity, make sure that the desired
graph is displayed on the Results screen.
To save your data, insert the Countess™ II USB drive (or equivalent) into an
1.
available USB port on the instrument (“Instrument exterior components“ on
page 11).
Note: The USB ports located in the front and the back of the instrument function
the same. However, the rst USB drive connected will be the preferred saving
location.
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Chapter 7
On the Results screen, press Save to go to the Save screen.
2.
To assign a name to your experiment, press the File name text eld. The alpha-
3.
numeric keypad opens.
Save results
Save count results
7
Enter the le name using the alpha-numeric keypad.
4.
To enter symbols, press the symbol (@%&) key. To return to the alpha-numeric
keypad, press ABC.
Press Enter to save the name and return to the Save screen.
5.
To return to the Save screen without saving the name, press the close buon.
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Chapter 7
7
Save count results
Save results
Select the desired mode to save your experiment (Result, Images, Data, Report).
6.
You can select an individual mode (e.g., Result only) or any combination of
modes (e.g., Result, Images, Data, and/or Report).
In the example below, Data and Report are selected.
By default, Result and Images are saved as JPEG les, and Report is saved as
7.
PDF.
To choose a dierent le format, press the le type buon. The Choose le type
screen opens.
Note: Data can only be saved as a CSV le.
Press to select the desired le type. Available options are JPEG, BMP, PNG, and
8.
TIFF.
46
After you make your selection, the instrument returns to the Save screen.
To return to the Save screen without changing the le format, press the close
buon.
Countess™ II FL automated cell counter User Guide
Page 47
Chapter 7 Save results
Press Save to save your experiment in the selected mode(s) in the USB drive.
9.
Press Close and then transfer the USB drive to the desired location.
10.
Report
7
Report
Report file
The Report function allows you to save a printer-friendly report of the results, graphs,
and images in the selected format (PDF, PNG, or JPEG).
You can create reports using the Report dropdown as described in “Save count
results“ on page 44.
Countess™ II FL automated cell counter User Guide
47
Page 48
Chapter 7 Save results
7
Report
Report from brightfield count
48
• The top section of the Report contains a table with the results as displayed on the
Countess™ Results screen, showing the concentration of the sample, and the percentage
and number for the total, Live, and Dead channels.
• Below the results table, the report contains the “number of cells vs. cell size” graph.
• Under the graph, the report contains the brightfield count image, with the live and dead
cells identified by the green and red circles, respectively.
• At the bottom, the report displays the profile information used to gate these images.
Countess™ II FL automated cell counter User Guide
Page 49
Report from fluorescence count
Chapter 7 Save results
Report
7
• The top section of the Report contains a table with the results as displayed on the
Countess™ Results screen, showing the concentration of the sample, and the percentage
and number of cells for the total, FL1, FL2, and FL1 + FL2 channels.
• Below the results table, the report contains the “number of cells vs. cell size” graph on the
left, and “number of cells vs. RFU (relative fluorescence units)” graph on the right.
• Under the graphs, the report contains the count images, with the brightfield image on the
left and the fluorescence images on the right.
In the brightfield image, the cells counted in the brightfield channel are identified by the
white “total count” circles.
In the fluorescence image (overlaid, if there are two channels), the cells counted are
identified by the circles with the same color as the fluorescence channel in which they
were counted (in two colors, if there are two cubes installed).
• At the bottom, the report displays the profile information used to gate these images.
Countess™ II FL automated cell counter User Guide
49
Page 50
8
Overview
Instrument settings
Instrument settings screen
To access the Instrument Seings screen, press the Instrument Seings
the Home page (“Turn ON the instrument“ on page 13).
In the Instrument Seings screen, you can:
• perform software update (“Software update“ on page 50)
• set the date and time (“Date/Time“ on page 52)
• change or install EVOS™ light cube (“Change light cube“ on page 54)
buon on
Software update
Guidelines for software update
50
• The USB drive used for transferring the software update le must be FAT32
formaed; verify this before proceeding. If necessary, reformat the USB drive to
FAT32 following the recommended procedure for your operating system.
Note: Reformaing the USB drive will result in the loss of all les. Back up the
les in the USB drive prior to reformaing.
• The software update le must be saved on the top level of the USB drive, not
within a folder or a subfolder.
• The software update le must be uncorrupted during transfer. Do not rename,
zip, or compress the software update le.
Countess™ II FL automated cell counter User Guide
Page 51
Update the
Countess™ II/II FL
software
Chapter 8 Instrument settings
Go to www.thermosher.com/countessupdate, and download the latest
1.
Countess™ II/II FL software version to your desktop.
Note: The software update le has a version-specic name followed by the
extension .lft (e.g., Countess™_II_v_1_0_202.lft for software version 1.0.202).
Copy the software update le onto the USB drive, making sure that it is saved on
2.
the top level and not hidden within a folder.
Insert the USB drive into one of the USB ports of the instrument (“Instrument
3.
exterior components“ on page 11).
Software update
8
Press the Instrument Seings
4.
instrument“ on page 13) to open the Instrument Seings screen (Chapter 8,
“Instrument seings“).
Select Software Update from the Instrument Seings menu. The instrument
5.
scans the USB drive for the latest software version.
When prompted, select Update Now.
6.
buon on the Home page (“Turn ON the
Once the update has completed, restart the instrument.
7.
Countess™ II FL automated cell counter User Guide
51
Page 52
Chapter 8 Instrument settings
8
Date/Time
Date/Time
Set the date and time
Press the Instrument Seings buon on the Home page (“Count
1.
parameters“ on page 15) to open the Instrument Seings screen.
Press Date/Time on the Instrument Seings menu to open the Date/Time screen.
2.
Select the Date format you wish to use.
3.
Press any Date text box (MM, DD, or YYYY) to open the Edit Date keypad.
4.
Using the keypad, enter the date into Month, Day, and Year text boxes, pressing
5.
Enter after each entry.
After you are nished entering the date, press the close buon to return to
6.
the Date/Time screen.
52
Countess™ II FL automated cell counter User Guide
Page 53
Chapter 8
Select the Time format you wish to use. Available options are 12 Hour and
7.
24 Hour formats.
Press any Time text box (Hours or Minutes) to open the Edit Time keypad.
8.
Instrument settings
Date/Time
8
Using the keypad, enter the time into Hours and Minutes text boxes, pressing
9.
Enter after each entry.
After you are nished entering the time, press the close buon to return to the
10.
Date/Time screen.
If you have selected the 12 Hour format, select AM or PM.
11.
Press Save to set the Time and Date and return to the Instrument Seings screen.
12.
Press Cancel to return to the Instrument Seings screen without saving your
changes.
Countess™ II FL automated cell counter User Guide
53
Page 54
Chapter 8 Instrument settings
8
Change light cube
Change light cube
Install or change EVOS™ light cube
The Countess™ II FL Automated Cell Counter can accommodate up to two EVOS
light cubes. Each user-interchangable, auto-congured EVOS™ light cube contains an
LED, collimating optics, and lters for uorescence applications. EVOS™ light cubes
do not come standard with the device and must be purchased separately (“EVOS
light cubes“ on page 64). To install or change a light cube:
Press the Instrument Seings buon on the Home page (“Turn ON the
1.
instrument“ on page 13) to open the Instrument Seings screen.
Press Change Light Cube. The instrument positions the light cube tray to enable
2.
light cube installation.
When prompted, power o the Countess™ II FL Automated Cell Counter using
3.
the power switch on the back of the instrument (“Instrument exterior
components“ on page 11).
Unplug the power cord from the Countess™ II FL Automated Cell Counter.
4.
Unlatch the back panel with the two captive ¼-turn fasteners (indicated by red
5.
arrows) that secure the back panel on the rear of the Countess™ II FL Automated
Cell Counter and remove the back panel.
™
™
54
Place the light cube into one of the empty slots in the back of the device.
6.
Countess™ II FL automated cell counter User Guide
Page 55
Chapter 8 Instrument settings
Using the tool provided on the inside of the back panel (Figure A), secure the
7.
light cube by tightening the two screws on the end of the cube (red arrows in
Figure B).
To remove a light cube, unscrew both screws that secure it to the instrument.
8.
Thread the light cube removal tool into the central hole in the cube (white arrows
9.
in Figure B) and gently pull the light cube out of the device.
Change light cube
8
Note: Always store the cube removal tool in the back panel for easy access.
Install the back panel and secure it in its place with both ¼-turn fasteners.
10.
Plug the power cord back into the Countess™ II FL Automated Cell Counter.
11.
Turn o the Countess™ II FL Automated Cell Counter by ipping the power
12.
switch on the back of the instrument to the ON position.
Countess™ II FL automated cell counter User Guide
55
Page 56
9
Instrument care
Maintenance
General guidelines for care
Power supply
• Use the appropriate cleaning solutions for each component, as indicated in the
cleaning procedures in “Clean the Countess™ II FL automated cell counter“ on
page 56.
• If liquid spills on the instrument, turn o the power immediately and wipe dry.
Always use the correct power supply. The power adaptor specications appear on the
serial number label (boom of the instrument) and in the Technical specications
section of this user guide (“Technical specications“ on page 63). Damage due to an
incompatible power adaptor is not covered by warranty.
CAUTION! Never disassemble or service the instrument yourself. Do not
remove any covers or parts that require the use of a tool to obtain access to
moving parts. Operators must be trained before being allowed to perform the
hazardous operation. Unauthorized repairs may damage the instrument or
alter its functionality, which may void your warranty. Contact your local
distributor to arrange for service.
IMPORTANT! If you have any doubt about the compatibility of decontamination or
cleaning agents with parts of the equipment or with material contained in it, contact
Technical Support () or your local distributor for information.
Clean the Countess™ II FL automated cell counter
Introduction
56
Clean the Countess™ II FL Automated Cell Counter periodically to prevent buildup of
dust and dirt that might reduce its performance and cause contamination.
CAUTION! To avoid electrical shock, always turn OFF the Countess
Automated Cell Counter and unplug the power cord before cleaning or
decontaminating the instrument.
CAUTION! All biological samples and materials that come into contact with
them have the potential to transmit infectious diseases and are considered
biohazardous. Follow all applicable local, state/provincial, and/or national
regulations. Wear appropriate protective eyewear, clothing, and gloves.
IMPORTANT! Using a cleaning or decontaminating method other than that specied
by the manufacturer may result in damage to the instrument.
Countess™ II FL automated cell counter User Guide
™
II FL
Page 57
Chapter 9 Maintenance
Set nominal focus
9
Clean the touchscreen
Clean the instrument case
Decontaminate the instrument
• Wipe the touch-screen of the Countess™ II FL Automated Cell Counter using a
soft, lint-free cloth moistened with an LCD cleaning solution. Do not apply
excessive force during cleaning. Wipe the touch-screen dry immediately after
cleaning.
• Ensure that the cleaning solution does not enter the power buon, the power
inlet, the slide port, or the USB ports.
• Never pour or spray any liquids directly on the instrument to avoid electrical
shock when the instrument is plugged in.
• Do not use abrasive cleaning solutions or material to prevent the touch-screen
from geing scratched.
• Wipe the instrument case of the Countess™ II FL Automated Cell Counter using a
soft, lint-free cloth moistened with distilled water. Wipe the instrument dry
immediately after cleaning.
• Ensure that water or other cleaning solutions do not enter the power buon, the
power inlet, the slide port, or the USB ports.
• Never pour or spray any liquids directly on the instrument to avoid electrical
shock when the instrument is plugged in.
• Wipe the instrument case of the Countess™ II FL Automated Cell Counter using a
soft, lint-free cloth moistened with 70% alcohol. Wipe the instrument dry
immediately after cleaning.
• Avoid using a bleach solution, because it may leave a residue of bleach crystals
on the instrument.
• Ensure that water or other cleaning solutions do not enter the power buon, the
power inlet, the slide port, or the USB ports.
• Never pour or spray any liquids directly on the instrument to avoid electrical
shock when the instrument is plugged in.
Set nominal focus
Overview
Countess™ II FL automated cell counter User Guide
Nominal focus is the Z-point (i.e., depth) around which the auto focus function
searches to provide ne focus to the sample.
The auto focus algorithm of the Countess™ II FL Automated Cell Counter is designed
to highlight the dierences between live and dead cells in the brighteld channel. The
optimal focus level is where the live cells have a light colored center and the dead
cells are dark throughout (see examples below).
57
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Chapter 9 Maintenance
9
Set nominal focus
Set nominal focus
To enable optimal auto focus functionality, you may need to initially rene the
brighteld focus by adjusting it manually and then seing the nominal focus. This
allows the auto focus function to have a set point from which to focus on the cells in
subsequent samples.
Prepare the sample by adding 10 µL of cell suspension to 10 µL of 0.4% trypan
1.
blue stain. Mix the sample mixture well by pipeing up and down a few times.
Load 10 µL of the sample mixture into the Countess™ Cell Counting Chamber
2.
Slide (“Load Countess™ chamber slide“ on page 19) or the Countess™ II FL
Reusable Slide (“Countess™ II FL reusable slide“ on page 21). Let the sample
mixture sele for 30 seconds to ensure a uniform focal plane.
Insert the sample slide into the slide port of the instrument (“Instrument exterior
3.
components“ on page 11), making sure that the side containing the sample is
inserted completely.
When the slide is inserted, the instrument automatically illuminates the sample,
4.
sets the intensity of the brighteld light source, and auto focuses on the cells.
58
To manually adjust the focus, press the Focus™ buon.
5.
Countess™ II FL automated cell counter User Guide
Page 59
Chapter 9 Maintenance
Set nominal focus
Use the Focus™ slider or the plus and minus buons to rene the brighteld
6.
focus.
Note: If needed, Zoom in on the image to adjust focus or lighting.
After nding the optimal focus, press Set to set the nominal focus.
7.
Once the focus has been set, the Set buon on the focus slider becomes
inactive, conrming that the focus seing has been stored.
9
Countess™ II FL automated cell counter User Guide
59
Page 60
A
Troubleshooting
Note: The software for the Countess™ II FL is updated regularly. If you are having
any issues with your experiments, rst check the website to see if a new software
version is available. You can download the most recent version of the software from
www.thermosher.com/countessupdate. You can also register your Countess™ II FL
instrument at www.thermosher.com/registercountess to be informed of any future
software updates.
Problem
Uneven screen illumination (screen is dark on one side,
but brighter on the other)
Autofocus does not seem to focus on the cells very well
Some cells appear on the image but are not included in
the count
Possible solutions
Reset the light cube tray by selecting Change Light Cube
on the Instrument Settings screen (Chapter 8,
“Instrument settings“).
• Make sure there are no bubbles or debris visible on
the screen that could interfere with the autofocus
and make it more difficult to get the sample in the
correct focal plane.
• Ideally, the live cells should have bright centers
compared to the dead cells, which are dark
throughout (“Set nominal focus“ on page 57).
• Setting the nominal focus will improve autofocus
consistency with future slides. To set the nominal
focus, see “Set nominal focus“ on page 57.
• For cell count and cell viability assays performed in
the brightfield, adjust the size, brightness, and
circularity gates for both live and dead cells to
include all of the cells in the count (“Gate count
results“ on page 27).
• For fluorescence assays, adjust the size, brightness,
circularity, and fluorescence intensity gates in all
available channels to include all of the cells in the
count (“Gate count results“ on page 37).
• After including all of the cells in the count, you can
narrow the count criteria, if you wish to exclude cells
of a certain size or certain brightness.
• When the gates are fully maximized, the CSV should
indicate 0–60 for cell size and 0–255 for brightness.
Images are very bright and washed out
Fluorescence is extremely bright and bleeding through
into other filters
60
Enable Auto Lighting from the Profiles menu, or decrease
the bright field light intensity before counting the cells.
Decrease the fluorescence light intensity before counting
the cells.
Countess™ II FL automated cell counter User Guide
Page 61
Appendix A Troubleshooting
Set nominal focus
Problem Possible solutions
A
Getting incorrect concentration for the Countess™ test
beads
Variable counts for the same sample of cells
Variable counts when performing replicate counts of the
same slide
• The beads can settle quickly in solution, which will
affect the concentration reading.
• Vortex the bead stock on high for a full 30 seconds to
resuspend, and add 10 µL of the bead suspension to
10 µL of trypan blue without delay.
• Pipet the bead and trypan blue mixture up and down
several times to make sure it is well mixed, and
immediately load 10 µL into the slide.
• If you are pipetting different samples from the same
cell sample, the variability could be due to pipetting
or mixing.
• Use recently calibrated pipettors and make sure that
the cells are well suspended by pipetting up and
down several times before adding trypan blue.
• Pipet the bead and trypan blue mixture up and down
several times to make sure it is well mixed, and load
10 µL into the slide without delay.
• If you are counting replicates of the exact same
slide, visually inspect that all cells are counted
correctly in the image.
• There may be a slightly different field of view each
time a slide is inserted. Depending on the
concentration and uniformity of the cells, this will
cause some variability when performing replicate
counts of the same slide, although it should be less
than 10%.
• When counting fewer cells, a small field of view
change for only a small number of cells can have a
larger affect. Count cells at a higher concentration to
reduce variability.
• Make sure that you do not shake or agitate the slide
between counts.
Countess™ II FL automated cell counter User Guide
61
Page 62
Appendix A Troubleshooting
A
Set nominal focus
Problem Possible solutions
Abnormally high percentage of dead cells or live cells
counted as dead
USB drive not recognized by the instrument
Unable to update the Countess™ software
• Ensure that the cells are focused correctly so that
live cells have bright centers and dead cells are dark
throughout (see “Set nominal focus“ on page 57). If
the cells are not well focused and look dark on the
screen, the Countess™ II FL will count them as dead
cells.
• If cells are well focused, have bright centers, and are
being counted as dead, confirm that they are within
the appropriate cell size range and try adjusting the
settings.
• If cells are exposed to trypan blue for a long period
of time, viability could be affected so slide should be
prepared and counted fresh each time for best
results.
• Gate out the debris using the size, brightness, and
circularity sliders.
• The USB drive must be FAT32 formatted to be
recognized by the instrument. If it is not, reformat
the USB drive to FAT32 (“Software update“ on
page 50).
• Try another correctly formatted USB drive.
• Make sure the USB drive is formatted to FAT32. If it
is not, reformat the USB drive to FAT32 before
transferring the files onto the USB drive for software
update.
• The update file must sit on the top level of the USB
drive, not within a folder or a subfolder.
• File cannot be renamed in any way.
• File cannot be zipped or compressed during
distribution. It must be uncorrupted during transfer
and have a .lft suffix.
• If needed, check that the USB port is functional by
testing a USB mouse.
62
Countess™ II FL automated cell counter User Guide
Page 63
B
Technical specifications
Product specifications
Physical characteristics
Technical
specifications
Instrument type:
Instrument dimensions: 9 (W) × 5½ (D) × 9 inches (H)
Weight: 8 lbs
Operating power: 100–240 VAC, 0.58 A MAX
Frequency: 50/60 Hz
Electrical input: 12 VDC, 2 A
Installation site: Indoor use only, Class A Environments (i.e.,
Operating temperature: 4°–40℃ (39°–104°F)
Operating humidity: <80% (non-condensing)
Processing time:
Sample concentration range: 1 × 104–1 × 107 cells/mL
Particle/cell diameter range: 4–60 µm (particles); 7–60 µm (cells)
Benchtop cell counter and suspension cell-
based assay platform
non-residential or light industrial);
Pollution degree 2.
∼15 seconds
Required sample volume: 10 µL
Firmware: Countess™ Automated Cell Counting
USB Drive : 4 Gigabytes
Optics
Analysis slide
Countess™ II FL automated cell counter User Guide
Optics:
Camera: 5 Mega pixels, 2.5× Optical Magnification
Material:
Dimensions: 25 mm (W) × 75 mm (D) × 1.7 mm (H)
Chamber volume: 10 µL
Platform Software
3 channels (brightfield and 2 slots for
EVOS™ LED light cubes)
Poly(methyl methacrylate) (PMMA)
63
Page 64
Appendix B Product specifications
B
EVOS™ light cubes
EVOS™ light cubes
LED illumination
EVOS™ light cubes
The Countess™ II FL Automated Cell Counter utilizes an adjustable intensity LED
light source provided by the proprietary, user-interchangeable LED light cube (see
“EVOS™ light cubes“ on page 64). Because the LED light source is as close as possible
to the objective, the number of optical elements in the channel is minimized. Highintensity illumination over a short channel increases the eciency of uorophore
excitation, providing beer detection of weak uorescent signals.
Each user-interchangable, auto-congured EVOS™ light cube contains an LED,
collimating optics, and lters. In addition to the brighteld channel dedicated to cell
count and cell viability assays using Trypan Blue, the Countess™ II FL Automated Cell
Counter can accommodate two uorescent light cubes for multiple-uorescence
research applications.
The following table lists some of the common uorescent and specialty EVOS™ light
cubes available from Thermo Fisher Scientic. For a complete list, go to
www.thermosher.com/evoslightcubes or contact Technical Support (). For
instructions on changing the LED light cubes, see “Change light cube“ on page 54.
Light cube
DAPI DAPI, Hoechst™, BFP
TagBFP TagBFP
CFP ECFP, Lucifer Yellow, Evans Blue
GFP GFP, Alexa Fluor™ 488, SYBR™ Green, FITC
YFP EYFP, acridine orange + DNA
RFP RFP, Alexa Fluor™ 546, Alexa Fluor™ 555, Alexa Fluor™ 568, Cy3™,
MitoTracker™ Orange, Rhodamine Red™, DsRed
Texas Red
Cy5 Cy5™, Alexa Fluor™ 647, Alexa Fluor™ 660, DRAQ5
Cy5.5 Cy™ 5.5, Alexa Fluor™ 660, Alexa Fluor™ 680, Alexa Fluor™ 700
Cy7 Cy™ 7, IRDye™ 800CW
™
Texas Red™, Alexa Fluor™ 568, Alexa Fluor™ 594, MitoTracker™ Red,
mCherry, Cy™3.5
Dye
™
64
Countess™ II FL automated cell counter User Guide
Page 65
Appendix B Product specifications
EVOS™ light cubes
Note: The EVOS™ light cubes are available only for the Countess™ II FL Automated
Cell Counter. The Countess™ II Automated Cell Counter uses only brighteld
illumination and does not support the EVOS™ light cubes.
B
Countess™ II FL automated cell counter User Guide
65
Page 66
Ordering information
C
Countess™ II FL automated cell counter and accessories
The following Countess™ II FL instruments and instrument accessories are available
from Thermo Fisher Scientic. For more information, visit www.thermosher.com or
contact Technical Support.
Countess™ II FL Automated Cell Counter 1 each AMQAF1000
Countess™ II power cord with four adapter cords 1 each AMEP4716
Countess™ II USB drive 1 each A25751
Countess™ II FL Light Cube Removal Tool 1 each AMEP4747
Countess™ II FL Disposable Slide Holder 1 each AMEP4745
Countess™ II FL Reusable Slide Holder 1 each AMEP4746
Accessory products
The following products can be used with the Countess™ II FL Automated Cell
Counter and are available separately from Thermo Fisher Scientic. For more
information, visit www.thermosher.com or contact Technical Support.
Countess™ Cell Counting Chamber Slides, 50 Slides (100
counts)
Countess™ Cell Counting Chamber Slides, 500 Slides (1000
Counts)
Product
Product
Quantity Cat. No.
Quantity Cat. No.
[1]
1 box
10 boxes
[1]
C10228
C10312
66
Countess™ Cell Counting Chamber Slides, 1250 Slides
(2500 Counts)
Countess™ Cell Counting Chamber Slides, 2500 Slides
(5000 Counts)
Countess™ Cell Counting Chamber Slides, 5000 Slides
(10,000 Counts)
Countess™ II FL Reusable Slide 1 each A25750
Countess™ II FL automated cell counter User Guide
25 boxes
50 boxes
100 boxes
[1]
[1]
[1]
C10313
C10314
C10315
Page 67
Appendix C Ordering information
Product Quantity Cat. No.
Countess™ Test Beads (1 × 106 beads/mL) 1 mL C10284
Trypan blue stain (0.4 %) 2 × 1 mL T10282
[1]
Each box of Countess™ Cell Counting Chamber Slides contains 50 slides and 2 × 1 mL vials of trypan blue
(0.4%), sufficient for 100 counts.
Accessory products
C
Countess™ II FL automated cell counter User Guide
67
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D
CSV file format, explained
CSV file format
Overview
Category
General A Number Sequential sample run number
Trypan Blue/Brightfield E Total Concentration Concentration of the entire sample
A comma-separated values (CSV) le stores tabular data (numbers and text) in plaintext form. Plain text means that the le is a sequence of characters, with no data that
has to be interpreted as binary numbers. A CSV le can be opened with any third
party software or spreadsheet program. The table below describes the categories of
the Countess™ II data saved as a CSV le and opened with a spreadsheet program.
Column Name Description
B File Name Name of file
C Date & Time Date and time of sample run
D Mode BF-Brightfield or FL-Fluorescence
F Total cells counted Total number of cells counted in the sample
G Live concentration Concentration of just the “live” portion of the
sample
H Live cells counted Total number of “live” cells counted
I Dead concentration Concentration of just the “dead” portion of the
sample
J Dead cells counted Total number of “dead” cells counted
K Viability (%) Percent viability of the sample based on trypan
blue staining
L Average size (um) Average cell size in microns
Fluorescence M Cube 1 name EVOS™ light cube name in the first (top) position
N Cube 1 concentration Concentration of cells showing fluorescence in the
first cube position
O Cube 1 (%) Percentage of the total cells in brightfield that
show fluorescence in the first cube position
P Cube 1 cells counted Total number of cells counted in the first cube
position
Q Cube 2 name EVOS™ light cube name in the second (bottom)
position
68
Countess™ II FL automated cell counter User Guide
Page 69
Appendix D CSV file format
CSV file format, explained
Category Column Name Description
Fluorescence R Cube 2 concentration Concentration of cells showing fluorescence in the
second cube position
S Cube 2 (%) Percentage of the total cells in brightfield that
show fluorescence in the second cube position
T Cube 2 cells counted Total number of cells counted in the second cube
position
U Cube 1+2 concentration Concentration of cells showing fluorescence in the
first and second cube positions combined
V Cube 1+2 (%) Percentage of the total cells in brightfield that
show fluorescence in the first and second cube
position combined
W Cube 1+2 cells counted Total number of cells counted in the first and
second cube position combined
General Details X Focus™ value Focal position number
D
Trypan Blue/Brightfield
Count Parameters
Fluorescence Count
Parameters
Y BF Light intensity Brightfield light intensity value from 0-100%
Z Live Size min Minimum size of “live” cells in microns
AA Live Size max Maximum size of “live” cells in microns
™
AB
AC Live Brightness max “Live” adjustment slider value for maximum
AD Live Circularity “Live” adjustment slider value for circularity
AE Dead Size min Minimum size of “dead” cells in microns
AF Dead Size max Maximum size of “dead” cells in microns
AG Dead Bright min “Dead” adjustment slider value for minimum
AH Dead Bright max “Dead” adjustment slider value for maximum
AI Dead Circ “Dead” adjustment slider value for circularity
AJ Cube 1 Light intensity First (top) light cube light intensity value from
Live Brightness min “Live” adjustment slider value for minimum
brightness
brightness
brightness
brightness
0-100%
AK Cube 2 Light intensity Second (bottom) light cube light intensity value
AL BF Size min Minimum size of “Brightfield” cells in microns
AM BF Size max Maximum size of “Brightfield” cells in microns
Countess™ II FL automated cell counter User Guide
from 0-100%
69
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Appendix D CSV file format
D
CSV file format, explained
Category Column Name Description
Fluorescence Count
Parameters
AN BF Brightness min “Brightfield” adjustment slider value for minimum
brightness
AO BF Brightness max “Brightfield” adjustment slider value for maximum
brightness
AP BF Circularity “Brightfield” adjustment slider value for circularity
AQ Cube 1 Brightness min First (top) light cube adjustment slider value for
minimum brightness
AR Cube 1 Brightness max First (top) light cube adjustment slider value for
maximum brightness
AS Cube 2 Brightness min Second (bottom) light cube adjustment slider value
for minimum brightness
AT Cube 2 Brightness max Second (bottom) light cube adjustment slider value
for maximum brightness
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Countess™ II FL automated cell counter User Guide
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E
Safety alert words
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specied
in the user documentation may result in personal injury or damage to the
instrument or device. Ensure that anyone using this product has received
instructions in general safety practices for laboratories and the safety
information provided in this document.
Before using an instrument or device, read and understand the safety
·
information provided in the user documentation provided by the
manufacturer of the instrument or device.
Before handling chemicals, read and understand all applicable Safety Data
·
Sheets (SDSs) and use appropriate personal protective equipment (gloves,
gowns, eye protection, and so on). To obtain SDSs, see the “Documentation
and Support” section in this document.
Four safety alert words appear in this document at points where you need to be aware
of relevant hazards. Each alert word—IMPORTANT, CAUTION, WARNING,
DANGER—implies a particular level of observation or action, as dened below:
IMPORTANT! – Provides information that is necessary for proper instrument
operation, accurate installation, or safe use of a chemical.
CAUTION! – Indicates a potentially hazardous situation that, if not avoided,
may result in minor or moderate injury. It may also be used to alert against
unsafe practices.
WARNING! – Indicates a potentially hazardous situation that, if not avoided,
could result in death or serious injury.
DANGER! – Indicates an imminently hazardous situation that, if not avoided,
will result in death or serious injury. This signal word is to be limited to the
most extreme situations.
Except for IMPORTANT! safety alerts, each safety alert word in this document
appears with an open triangle gure that contains a hazard symbol. These hazard
symbols are identical to the hazard symbols that are axed to the instruments (see
Safety symbols in Appendix E).
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Appendix E Safety
E
Electrical symbols
Electrical symbols
The following table describes the electrical symbols that may be displayed.
Symbol Description
Indicates the On position of the main power switch.
Indicates the Off position of the main power switch.
Indicates a standby switch by which the instrument is switched on to the
Standby condition. Hazardous voltage may be present if this switch is on
standby.
Indicates the On/Off position of a push-push main power switch.
Indicates a terminal that may be connected to the signal ground reference of
another instrument. This is not a protected ground terminal.
Safety symbols
Indicates a protective grounding terminal that must be connected to earth
ground before any other electrical connections are made to the instrument.
Indicates a terminal that can receive or supply alternating current or
voltage.
Indicates a terminal that can receive or supply alternating or direct current
or voltage.
The following table describes the safety symbols that may be displayed. Each symbol
may appear by itself or in combination with text that explains the relevant hazard (see
“Safety labels on instruments”). These safety symbols may also appear next to
DANGERS, WARNINGS, and CAUTIONS that occur in the text of this and other
product-support documents.
Symbol
Caution, risk of danger. Consult the manual for further safety information.
Description
72
Caution, risk of electrical shock.
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Symbol Description
Caution, hot surface or other high-temperature hazard.
Caution, laser.
Caution, moving parts.
Caution, potential biohazard.
Appendix E Safety
Environmental symbols
E
Environmental symbols
The following symbol applies to all Thermo Fisher Scientic electrical and electronic
products placed on the European market after August 13, 2005.
Symbol
Caution, ultraviolet light.
Description
Do not dispose of this product as unsorted municipal waste. Follow local
municipal waste ordinances for proper disposal provisions to reduce the
environmental impact of waste electrical and electronic equipment (WEEE).
European Union customers:
Call your Customer Service representative for equipment pick-up and
recycling. See www.thermofisher.com for a list of customer service offices in
the European Union.
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Appendix E Safety
E
Safety labels on instruments
Safety labels on instruments
The following CAUTION, WARNING, and DANGER statements may be displayed
Thermo Fisher Scientic instruments in combination with the safety symbols
described in the preceding section.
Symbol English Français
CAUTION! Hazardous chemicals. Read the Safety
Data Sheets (SDSs) before handling.
CAUTION! Hazardous waste. Refer to SDS(s) and
local regulations for handling and disposal.
DANGER! High voltage. DANGER! Haute tension.
WARNING! To reduce the chance of electrical
shock, do not remove covers that require tool
access. No user-serviceable parts are inside. Refer
servicing to Thermo Fisher Scientific qualified
service personnel.
DANGER! Class 3B visible and/or invisible laser
radiation present when open. Avoid exposure to
beam.
CAUTION! Moving parts. Crush/pinch hazard. ATTENTION! Pièces en mouvement, risque de
ATTENTION! Produits chimiques dangereux. Lire
les fiches techniques de sûreté de matériels avant
toute manipulation de produits.
ATTENTION! Déchets dangereux. Lire les fiches
techniques de sûreté de matériels et la régulation
locale associées à la manipulation et l’élimination
des déchets.
AVERTISSEMENT! Pour éviter les risques
d’électrocution, ne pas retirer les capots dont
l’ouverture nécessite l’utilisation d’outils.
L’instrument ne contient aucune pièce réparable
par l’utilisateur. Toute intervention doit être
effectuée par le personnel de service qualifié
venant de Thermo Fisher Scientific.
DANGER! Rayonnement visible ou invisible d’un
faisceau laser de Classe 3B en cas d’ouverture.
Evitez toute exposition au faisceau.
pincement et/ou d’écrasement.
General instrument safety
WARNING! PHYSICAL INJURY HAZARD. Use this product only as specied
in this document. Using this instrument in a manner not specied by Thermo
Fisher Scientic may result in personal injury or damage to the instrument.
Operating the instrument
74
Ensure that anyone who operates the instrument has:
• Received instructions in both general safety practices for laboratories and specic
safety practices for the instrument.
• Read and understood all applicable Safety Data Sheets (SDSs).
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Appendix E Safety
General instrument safety
E
Safety precautions
• Do not install the instrument in heavy humidity, such as a greenhouse or an
incubator, to avoid a danger of electric shock. If water or other material enters the
instrument, the adaptor, or power inlet, disconnect the power cord and contact a
service person.
• Do not press the main plug or power cord with wet hands.
• Always ensure that the power supply input voltage matches the voltage available
in your location.
• Do not install the instrument on a slant or a place prone to vibrations, which
induces the risk of instrument malfunction or damage of the instrument.
• Never insert any objects into the air vents of the instrument as this could result in
electrical shock, personal injury, and equipment damage.
• Plug the power cord rmly into the wall outlet and the instrument.
• To avoid potential shock hazard, make sure that the power cord is properly
grounded.
• Be sure to position the equipment such that it is easy to disconnect the
instrument.
• Turn o the instrument before unplugging the power cord and/or moving the
instrument.
• If the instrument is broken or dropped, disconnect the power cord and contact a
service person. Do not disassemble the instrument.
• Use only authorized accessories (adaptor, power cord, and USB drive).
• If the instrument emits smoke, disconnect the power cord from the wall outlet
and contact a service person.
Cleaning or decontaminating the instrument
Removing covers or parts of the instrument
CAUTION! Using cleaning or decontamination methods other than those
recommended by the manufacturer may compromise the safety or quality of
the instrument.
CAUTION! PHYSICAL INJURY HAZARD The instrument is to be serviced
only by trained personnel or vendor specied in the user guide.
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Appendix E Safety
E
Chemical safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,
ensure laboratory personnel read and practice the general safety guidelines for
chemical usage, storage, and waste provided below. Consult the relevant SDS
for specic precautions and instructions:
Read and understand the Safety Data Sheets (SDSs) provided by the
·
chemical manufacturer before you store, handle, or work with any chemicals
or hazardous materials. To obtain SDSs, see the "Documentation and
Support" section in this document.
Minimize contact with chemicals. Wear appropriate personal protective
·
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing).
Minimize the inhalation of chemicals. Do not leave chemical containers open.
·
Use only with sucient ventilation (for example, fume hood).
Check regularly for chemical leaks or spills. If a leak or spill occurs, follow
·
the manufacturer cleanup procedures as recommended in the SDS.
Handle chemical wastes in a fume hood.
·
Ensure use of primary and secondary waste containers. (A primary waste
·
container holds the immediate waste. A secondary container contains spills
or leaks from the primary container. Both containers must be compatible
with the waste material and meet federal, state, and local requirements for
container storage.)
After emptying a waste container, seal it with the cap provided.
·
Characterize (by analysis if needed) the waste generated by the particular
·
applications, reagents, and substrates used in your laboratory.
Ensure that the waste is stored, transferred, transported, and disposed of
·
according to all local, state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require special
·
handling, and disposal limitations may apply.
WARNING! HAZARDOUS WASTE (from instruments). Waste produced by
the instrument is potentially hazardous. Follow the guidelines noted in the
preceding General Chemical Handling warning.
WARNING! 4L Reagent and Waste Bole Safety. Four-liter reagent and waste
boles can crack and leak. Each 4-liter bole should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in
the upright position.
Chemical waste safety
Chemical waste hazard
76
CAUTION!
local regulations for handling and disposal.
HAZARDOUS WASTE. Refer to Safety Data Sheets (SDSs) and
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Appendix E Safety
Electrical safety
E
Chemical waste safety guidelines
Waste disposal
To minimize the hazards of chemical waste:
• Read and understand the Safety Data Sheets (SDSs) provided by the
manufacturers of the chemicals in the waste container before you store, handle, or
dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks from
the primary container. Both containers must be compatible with the waste
material and meet federal, state, and local requirements for container storage.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
only with adequate ventilation (for example, fume hood). For additional safety
guidelines, consult the SDS.
• Handle chemical wastes in a fume hood.
• After emptying the waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bole in accordance with
good laboratory practices and local, state/provincial, or national environmental
and health regulations.
If potentially hazardous waste is generated when you operate the instrument, you
must:
• Characterize (by analysis, if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and disposed
of according to all local, state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require special handling,
and disposal limitations may apply.
Electrical safety
Part
General DANGER! ELECTRICAL SHOCK HAZARD. Severe electrical shock
Fuses WARNING! FIRE HAZARD. For continued protection against the
Countess™ II FL automated cell counter User Guide
Symbol Description
can result from operating the Countess™ II Automated Cell
Counter or Countess™ II FL Automated Cell Counter without its
instrument panels in place. Do not remove instrument panels.
High-voltage contacts are exposed when instrument panels are
removed from the instrument.
risk of fire, replace fuses only with fuses of the type and rating
specified for the instrument.
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Appendix E Safety
E
Biological hazard safety
Part
Power DANGER! ELECTRICAL HAZARD. Grounding circuit continuity is
Overvoltage rating The Countess™ II Automated Cell Counter and Countess™ II FL
Biological hazard safety
WARNING! Biological samples such as tissues, body uids, and blood of
humans and other animals have the potential to transmit infectious diseases.
Follow all applicable local, state/provincial, and/or national regulations. Wear
appropriate protective eyewear, clothing, and gloves. Read and follow the
guidelines in these publications.
Symbol Description
vital for the safe operation of equipment. Never operate equipment
with the grounding conductor disconnected.
DANGER! ELECTRICAL HAZARD. Use properly configured and
approved line cords for the voltage supply in your facility.
DANGER! ELECTRICAL HAZARD. Plug the system into a properly
grounded receptacle with adequate current capacity.
Automated Cell Counter have an installation (overvoltage)
category of II, and are classified as portable equipment.
ATTENTION! BIOHAZARD. Les échantillons biologiques tels que les tissus, les
uides corporels et le sang des humains et d’autres animaux ont la possibilité de
transmere des maladies infectieuses. Suivre tous les règlements municipaux,
provinciaux/provincial et / ou nationales en vigueur. Porter des lunees de protection
approprié, des vêtements et des gants.
In the U.S.:
• U.S. Department of Health and Human Services, Biosafety in Microbiological and
Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112,
Revised December 2009; found at: www.cdc.gov/labs/pdf/CDC-
BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf
• Occupational Safety and Health Standards, Bloodborne Pathogens (29
CFR§1910.1030; www.access.gpo.gov/nara/cfr/waisidx_01/29cfr1910a_01.html)
• Your company’s/institution’s Biosafety Program protocols for working
with/handling potentially infectious materials.
• Additional information about biohazard guidelines is available at: www.cdc.gov
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Safety and electromagnetic compatibility (EMC) standards
Appendix E Safety
In the EU:
• Check your local guidelines and legislation on biohazard and biosafety
precaution, and the best practices published in the World Health Organisation
(WHO) Laboratory Biosafety Manual, third edition
www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004
_11/en/
Safety and electromagnetic compatibility (EMC) standards
E
Symbol
Description
U.S. and Canadian safety standards.
The CSA C/US Mark signifies that the product meets applicable U.S. and Canadian standards,
including those from CSA, CSA America, ANSI, ASME, ASSE, ASTM, NSF and UL.
European safety and EMC standards.
The CE Mark symbolizes that the product conforms to all applicable European Community
provisions for which this marking is required. Operation of the instrument is subject to the
conditions described in this manual.
The protection provided by the instrument may be impaired if the instrument is used in a manner
not specified by Thermo Fisher Scientific.
Australian EMC standards
The C-Tick Mark indicates conformity with Australian and New Zealand standards for
electromagnetic compatibility.
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Documentation and support
Customer and technical support
Visit thermosher.com/support for the latest service and support information.
• Worldwide contact telephone numbers
• Product support information
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support
• Product documentation
– User guides, manuals, and protocols
– Certicates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers,
contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale at
www.thermosher.com/us/en/home/global/terms-and-conditions.html. If you have
any questions, please contact Life Technologies at www.thermosher.com/support.
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thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com
7 June 2019
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