Thermo Fisher Scientific Countess User Manual

Countess™ 3 FL Automated Cell Counter

USER GUIDE

Catalog Number AMQAF2000

Publication Number MAN0019567

Revision B.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. MAN0019567

Revision Date Description

B.0 28 January 2021 Add component to Contents Table. Update Technical speciﬁcation.

A.0 09 November 2020 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.

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CHAPTER 1 Product information .................................................. 7

Contents ....................................................................... 7

Product description ............................................................. 8

Upon receiving the instrument .................................................... 8

Exterior instrument parts ......................................................... 9

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CHAPTER 2 Installation ........................................................... 10

Operating environment ......................................................... 10

Install the instrument ........................................................... 10

Turn ON the instrument ......................................................... 10

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CHAPTER 3 Templates ............................................................ 14

Templates screen .............................................................. 14

Template functions ............................................................. 15

Count parameters .............................................................. 15

Load a template ............................................................... 16

Create a template .............................................................. 16

Edit a template ................................................................ 16

Import a template .............................................................. 17

Export a template .............................................................. 18

Delete a template .............................................................. 19

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CHAPTER 4 Slide preparation and operation ................................... 20

Recommendations ............................................................. 20

Load Countess™ Cell Counting Chamber Slide ..................................... 20

Load the Countess™ Reusable Slide .............................................. 21

Countess

™

3 FL Automated Cell Counter User Guide

Contents

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CHAPTER 5 Cell count and cell viability assays ................................ 24

Count cells in brightﬁeld ........................................................ 24

Capture and count ......................................................... 24

Next steps ................................................................ 25

View results ................................................................... 26

Results screen for brightﬁeld ................................................ 26

Identify objects counted ........................................................ 26

Results screen ............................................................ 26

Identify cells counted in cell count and cell viability assays ...................... 27

Histogram count results ......................................................... 28

View histogram ............................................................ 28

Gate count results ............................................................. 28

Gating screen ............................................................. 28

Gate count results ......................................................... 29

Save as new template .......................................................... 29

Edit and save a new template ............................................... 29

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CHAPTER 6 Fluorescence assays ............................................... 30

Count cell ﬂuorescence ......................................................... 30

Overview ................................................................. 30

Count procedure .......................................................... 30

Next steps ................................................................ 33

View results ................................................................... 33

Results screen for cell ﬂuorescence assays ................................... 33

Identify objects counted ........................................................ 34

Identify cells counted in ﬂuorescence assays ................................. 34

Histogram count results ......................................................... 34

View histogram for cell ﬂuorescence assays ................................... 34

Gate count results ............................................................. 36

Gating screen ............................................................. 36

Gate count results ......................................................... 36

Save as new template .......................................................... 37

Edit and save a new template ............................................... 37

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CHAPTER 7 Calculators .......................................................... 38

Pre-Dilution Calculator .......................................................... 38

Cell Splitting Calculator ......................................................... 39

Countess™ 3 FL Automated Cell Counter User Guide

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CHAPTER 8 Save results ......................................................... 41

Save screen ................................................................... 41

Save procedure ................................................................ 41

Report ........................................................................ 44

Report ﬁle ................................................................ 44

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CHAPTER 9 Settings overview ................................................... 46

Settings screen ................................................................ 46

Connect to a wireless network ................................................... 46

Software update ............................................................... 47

Guidelines for software update .............................................. 47

Update the Countess™ 3/3 FL Cell Counter software ........................... 47

Set the date and time ........................................................... 48

Change your cloud region ....................................................... 49

Contents

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CHAPTER 10 Change EVOS™ light cubes ...................................... 51

Install or change an EVOS™ light cube ............................................ 51

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CHAPTER 11 Maintenance ....................................................... 53

Instrument care ................................................................ 53

General guidelines for care .................................................. 53

Power supply ............................................................. 53

Clean the cell counter .......................................................... 54

Introduction ............................................................... 54

Clean the touchscreen ..................................................... 54

Clean the instrument case .................................................. 54

Decontaminate the instrument ............................................... 55

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APPENDIX A Troubleshooting .................................................... 56

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APPENDIX B Technical speciﬁcations ........................................... 59

Physical characteristics ......................................................... 59

Technical speciﬁcations ......................................................... 59

Optics ........................................................................ 59

Analysis slide .................................................................. 60

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APPENDIX C EVOS light cubes .................................................. 61

LED illumination ............................................................... 61

EVOS™ light cubes ............................................................. 61

Countess™ 3 FL Automated Cell Counter User Guide

Contents

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APPENDIX D Countess™ 3 FL Automated Cell Counter and accessories ... 63

Accessory products ............................................................ 63

APPENDIX E CSV ﬁle format deﬁnition .......................................... 65

Overview ...................................................................... 65

APPENDIX F Safety ............................................................... 69

Chemical safety ................................................................ 70

Biological hazard safety ......................................................... 71

APPENDIX G Documentation and support ...................................... 72

Customer and technical support ................................................. 72

Limited product warranty ........................................................ 72

Countess™ 3 FL Automated Cell Counter User Guide

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

The Countess™ 3 FL Automated Cell Counter is shipped with the components listed below.

Countess™ 3 FL Automated Cell Counter 1 each

Power cord with 4 adapters (for U.S./Canada/Taiwan/Japan, Europe, or UK) 1 each

Countess™ Cell Counting Chamber Slides (50 slides/box) 1 box

Product information

Component Quantity

Countess™ 3 FL Disposable Slide Holder 1 each

Countess™ 3 FL Reusable Slide Holder 1 each

Countess™ 3 FL Light Cube Removal Tool 1 each

Countess™ 3 USB Drive 1 each

Countess™ 3 FL Automated Cell Counter Quick Reference Card 1 each

Wi-Fi Dongle 1 each

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 1 Product information

Product description

The Countess™ 3 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 ﬂuorescently labeled cells or trypan blue stained samples in suspension.

• The Countess™ 3 FL Automated Cell Counter oers an intuitive user interface with 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™ 3 FL Reusable Slides (page 19) . Each chamber slide contains two enclosed chambers to hold the sample to allow you to measure two dierent samples or perform replicates of the same sample.

• The instrument takes 10-20 seconds per sample for a typical cell count in the brightﬁeld channel and is compatible with a wide variety of eukaryotic cells. In addition to cell count and viability, the Countess™ 3 FL Automated Cell Counter also provides information on cell size.

• In addition to the brightﬁeld channel, the Countess™ 3 FL Automated Cell Counter can accommodate two interchangeable EVOS™ ﬂuorescent light cubes (page 64) , enabling it to be used for multiple-ﬂuorescence research applications.

• When equipped with EVOS™ light cubes, the Countess™ 3 FL Automated Cell Counter can be used to perform ﬂuorescence assays for cells in suspension, including simultaneous counts of cells stained with two dierent ﬂuorescent dyes, GFP and RFP expression, apoptosis, and cell viability (live, dead, and total cells).

Upon receiving the instrument

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 ﬁled with the carrier; the warranty does not cover in-transit damage.

See “Install the instrument” on page 10 for instructions on installing the instrument.

Register your instrument

Visit www.thermoﬁsher.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 notiﬁcations of software upgrades and information on new assays for use with the Countess™ cell counter.

Countess™ 3 FL Automated Cell Counter User Guide

Exterior instrument parts

Touchscreen display: The 7-inch capacitive display is the main user interface of the Countess™ 3 FL Automated Cell Counter. It contains the buttons for all instrument functions and displays data from the cell count.

Front USB port: Allows 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-, exFAT- or NTFS-formatted USB drive for data transfer. If desired, you can plug in a USB mouse into the rear USB port for instrument control.

Slide port: Used to insert the analysis slide containing the sample into the counter. The Countess™ 3 FL Automated Cell Counter accepts both the disposable Countess™ Cell Counting Chamber Slides

and the glass Countess™ 3 FL Reusable Slides via interchangeable, slide-speciﬁc carriers. For more information, see Chapter 4, “Slide preparation and operation”.

Back panel: 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.

Power switch: 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.

EVOS™ light cubes: Allow the Countess™ 3 FL Automated Cell Counter to analyze ﬂuorescently labeled samples. Can accommodate two ﬂuorescent light cubes. For more information, see “EVOS™ light cubes” on page 61.

Rear USB port: An additional USB port to perform functions such as plugging in a USB mouse for instrument control.

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.

Chapter 1 Product information

Exterior instrument parts

Countess™ 3 FL Automated Cell Counter User Guide

Operating environment

•

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 aect the image quality.

•

Operating temperature range: 4°–32°C (40°–90°F).

•

Relative humidity range: <80%.

IMPORTANT! Do not position the instrument so that it is dicult to turn o the main power switch

located on the back of the instrument.

In case of an instrument malfunction, turn the main power switch to the OFF position and disconnect the instrument from the wall outlet.

Installation

Install the instrument

Unpack the instrument and place the instrument on a ﬂat, level, dry surface.

Remove the thin plastic protector ﬁlm from the touch-screen display.

Plug one end of the power cord appropriate for your region into the instrument.

Plug the power cord into the electrical outlet. Be sure to use only the power cord supplied with your instrument. Powering the instrument with an unapproved power cord may damage the instrument.

Turn ON the instrument

After unboxing, upon switching on the instrument for the ﬁrst time, it will run through a series of set-up screens.

• Read the End User License Agreement and press Agree to proceed with start up.

• Set the Date and Time using the buttons (see “Set the date and time” on page 48). Press Next.

• Upon completion, press Done. You will proceed to the Home screen and can begin using the instrument as described below.

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 2 Installation

Sign in to the instrument with a new Connect account

Turn on the instrument by ﬂipping the power switch on the back of the instrument to the ON position. See “Exterior instrument parts” on page 9. The instrument will walk you through an initial Out-of-Box Experience to set up the instrument for

ﬁrst use.

From the Home screen, you can proceed immediately to the assays by inserting a slide. See Chapter 5, “Cell count and cell viability assays”.

Alternatively, you can change or add a template in step 3 below or change instrument settings in step 4 below.

To change the current template or add a new template to the instrument, press

Templates allows you to create customized count preferences (i.e., gate counts based on cell size, brightness, circularity and/or ﬂuorescence intensity). See “Load a template” on page 16.

To change instrument settings, press

Settings allows you to update the Countess™ 3 FL instrument software, customize instrument settings, view instrument details, export error logs, and change up to two EVOS™ light cubes. See “Settings screen” on page 46.

Settings .

Templates .

Note: A Wi-Fi adapter needs to be installed and a Thermo Fisher Connect account is required for

linking. See http://thermoﬁsher.com/connect for details on using Connect.

Press Sign in from the Home screen.

Press Link account.

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 2

Installation

Choose a method to connect.

• Press Mobile device. Scan the QR code on your instrument using the Instrument Connect app on your mobile device. Follow the steps shown on the page.

• Press PC to obtain a linking code to enter online in your Thermo Fisher Connect account.

A completion screen will show. Press Done. You then return to the Home screen and are logged into your account.

Once set up, you will then only need to sign in to your instrument account using your Username and PIN.

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 2 Installation

Sign in to the instrument with an existing Connect account

Press Sign in from the Home screen.

Select your Username from the drop-down box. Enter your PIN.

Press Sign in.

Countess™ 3 FL Automated Cell Counter User Guide

Templates screen

Templates allows you to create and save customized templates. Each custom template deﬁnes the count parameters (size, brightness, circularity, and ﬂuorescence intensity) for a consistent and streamlined workﬂow.

Templates

• Templates can be accessed from the Home and Gating screens.

• The currently selected template is displayed at the bottom of the Home screen.

• Automatic instrument functions and count parameters are deﬁned in the Edit template screen. See “Create a template” on page 16 or “Edit a template” on page 16.

• The Default template contains default count settings and cannot be edited.

• The count parameters speciﬁed in the selected template are applied to all new cell counts.

• Templates can be sorted by pressing on the Name, Channels, or Last Modiﬁed headers.

• If you have already performed a count, loading a new template from the Results screen applies the count preferences to the current count results (total cells, viability, etc.) and to all new counts.

Countess™ 3 FL Automated Cell Counter User Guide

Template functions

The Actions screen allows you to edit, import, export, and delete templates. Edit can be used to modify the currently selected template, while Import, Export, and Delete can be used on multiple templates.

Count parameters

Chapter 3 Templates

Template functions

Count parameters are adjusted in the Edit template screen using the Gating sliders. Moving the top and bottom gate boundaries adjusts the range of size, brightness, and circularity parameters.

Size Gating : Moving the top gate down removes larger objects; moving it up includes larger objects. Moving the bottom gate up removes smaller objects; moving it down includes smaller objects.

Brightness Gating: Moving the top gate down removes brighter objects; moving it up includes brighter objects. Moving the bottom gate up removes dimmer objects; moving it down includes dimmer objects.

Cicularity Gating: Moving the top gate down removes more circular objects; moving it up includes more circular objects. Moving the bottom gate up removes less circular objects; moving it down includes less circular objects.

Gating Boundary Sliders

Live or Dead can be selected only on a brightﬁeld count. For a ﬂuorescence count, select the individual

channels.

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 3

Load a template

Templates

Load a template

Press Templates.

Press the desired template to select, then press Load.

To return to the previous screen without loading the new template, press Cancel or The instrument will keep the saved template, but will return to the previous screen without loading it.

Create a template

Press Templates.

Press Create. To assign a name to the new template or to change the name of the existing template, press the Template name text box to open the keypad.

Back.

Type in the desired template name. 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 New Template screen. Press to return to the New Template screen without saving the name.

Press Save to save the new template. Press Cancel or without saving.

Edit a template

Press

Select an existing template, then press Actions.

Note: The default template contains default count settings and cannot be edited.

Templates.

Back to return to the Templates screen

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 3

Import a template

Press Edit.

The Edit screen for the selected template opens.

Press the appropriate channel (BF or other dye) and edit the desired parameters (see “Count parameters” on page 15) to create a custom template.

Press Save.

Templates

From the Templates screen, select the desired template and press Load to use that template to read the sample.

Import a template

Press

Press Actions.

Press Import.

Press the import location Cloud or USB.

Countess™ 3 FL Automated Cell Counter User Guide

Templates.

For Cloud, press Sign in to log in to your Thermo Fisher Connect account. If the Cloud is not active and disconnected, press Network settings to connect to a network, then press Back to try and sign in again.

Chapter 3

Export a template

Templates

For USB, insert your USB drive into the slot on the front of the instrument.

Press Next.

Select one or more templates from the list.

Press Import.

Once complete, press Done.

Export a template

Press Templates.

Press Actions.

Press Export.

Select one or more templates from the list.

Press Next.

Press the export location Cloud or USB.

Countess™ 3 FL Automated Cell Counter User Guide

For USB, insert your USB drive into the slot on the front of the instrument.

Press Export.

Once complete, press Done.

Delete a template

Chapter 3

Delete a template

Templates

Press Templates.

Press Actions.

Press Delete.

Select one or more templates from the list.

Press Delete. The system will prompt you to conﬁrm the deletion, then press Delete again.

Once complete, press Done.

Countess™ 3 FL Automated Cell Counter User Guide

Slide preparation and operation

The Countess™ 3 and 3 FL cell counters accept disposable Countess™ Cell Counting Chamber Slides and glass Countess™ Reusable Slides on interchangeable, slide-speciﬁc carriers.

Recommendations

To obtain the best results, follow these recommendations:

•

Ensure that the cell sample is homogeneously mixed.

•

The measurement range extends from 1 × 104 to 1 × 107 cells/mL, but the optimal range is 1 × 10 to 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.

Load Countess™ Cell Counting Chamber Slide

Prepare the sample by adding 10 µL of your cell suspension to 10 µL of 0.4% trypan blue stain (for brightﬁeld only). Mix the sample mixture well by pipetting it up and down a few times.

Gently pipet 10 µL of the sample into the half moon-shaped sample loading area. The sample is loaded into the chamber through capillary action.

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 4 Slide preparation and operation

Load the Countess™ Reusable Slide

Let the sample mixture settle in the chamber for 30 seconds, and then insert the slide into the slide adapter. You will hear a soft click once the slide is pushed in correctly.

Note: The instrument reads one side of the slide at a time, so insert the sides appropriately.

Note: The instrument is shipped with the slide adapter already installed.

Use the touchscreen to run the sample count. See Chapter 5, “Cell count and cell viability assays”.

To remove the slide, push the slide gently into the instrument until it clicks and the slide springs 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.

(Optional) To remove the slide adapter, gently squeeze the tabs and pull the adapter completely out of the instrument.

Load the Countess™ Reusable Slide

Before loading your sample into the Countess™ Reusable Slide, place a cover slip on the counting chamber, making sure the cover slip is clean and free of grease.

Prepare the sample by adding 10 µL of your cell suspension to 10 µL of 0.4% trypan blue stain (for brightﬁeld only). Mix the sample mixture well by pipetting it up and down a few times. For a ﬂuorescence sample, only add 10 µL of your sample neat.

Countess™ 3 FL Automated Cell Counter User Guide

Chapter 4 Slide preparation and operation

Load the Countess™ Reusable Slide

Gently pipet 10 µL of the sample into the sample inlet, allowing capillary action to draw the sample into the counting chamber. A properly loaded counting chamber should have a thin, even ﬁlm of ﬂuid under the cover slip.

Note: Each chamber in the Countess™ Cell Counting Chamber Slide or the Countess™ 3 FL Reusable Slide has a 10‑µL sample capacity. Do not overﬁll the slide chambers.

To use the Countess™ Reusable Slide, unlatch the back panel of the Countess™ 3 FL Automated Cell Counter with the two captive ¼-turn fasteners that secure the back panel on the rear of the instrument.

Countess™ 3 FL Automated Cell Counter User Guide

Remove the reusable slide holder from inside of the back panel.

Load the reusable glass slide into the reusable slide holder.

Chapter 4

Slide preparation and operation

Load the Countess™ Reusable Slide

Insert the reusable slide holder containing the slide into the slide port and gently push into the instrument until it clicks into place.

Use the touchscreen to run the sample count. See Chapter 5, “Cell count and cell viability assays”.

To remove the reusable slide holder, push the slide gently into the instrument until it clicks and a spring pushes the slide out. Grasp the slide and pull it out the rest of the way.

10.

(Optional): To count the second sample present on the reusable slide, simply remove the slide from the holder, rotate, and reinsert the slide into the holder so that the second sample is aligned with the sample viewing hole.

11.

After using the Countess™ Reusable Slide, rinse the glass slide and cover slip with water, and then clean with 70% ethanol. Use Kimwipes™ laboratory tissues to clean and dry the slides, as needed.

12.

Store the reusable slide holder again in the panel on the back of the instrument.

Countess™ 3 FL Automated Cell Counter User Guide

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