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.
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
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
3
Contents
■
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
4
Countess™ II FL automated cell counter User Guide
■
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
5
Contents
■
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
6
Countess™ II FL automated cell counter User Guide
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
7
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).
8
Countess™ II FL automated cell counter User Guide
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.
9
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.
10
Countess™ II FL automated cell counter User Guide
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
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.
12
Countess™ II FL automated cell counter User Guide
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
13
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
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
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).
16
Countess™ II FL automated cell counter User Guide
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
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
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
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
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.
Countess™ II FL automated cell counter User Guide
21
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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Countess™ II FL automated cell counter User Guide
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.
Countess™ II FL automated cell counter User Guide
23
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.
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Countess™ II FL automated cell counter User Guide
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.
Countess™ II FL automated cell counter User Guide
25
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