ThermoFisher Scientific invitrogen Countess 3 User manual

For Research Use Only. Not for use in diagnostic procedures.
Countess™3 Automated Cell Counter
USER GUIDE
Catalog Number AMQAX2000
Publication Number MAN0019566
Revision E.0

Life Technologies Corporation | 22025 20th Ave SE Ste. 100 | Bothell, Washington 98021 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
Revision history:MAN0019566 E.0 (English)
Revision Date Description
E.0 18 September
2023 Update latest draft of product manuals with latest screens and user workflow for SW#3.
D.0 16 September
2022 Adding network-drive saving and Operational Qualification (OQ) functionalities.
C.0 9 June 2021 Changing "Templates" functions to "Protocols". Adding additional settings in protocol editing/creation to include
Setup, Calculators, and Save.
B.0 28 January 2021 Add component to Contents table. Update technical specifications.
A.0 9 November 2020 New user guide for Countess™3 Automated Cell Counter.
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.
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.
©2020-2023 Thermo Fisher Scientific Inc. All rights reserved.

Contents
■CHAPTER1Productinformation .................................................. 6
Contents ....................................................................... 6
Productdescription ............................................................. 6
Upon receiving theinstrument .................................................... 7
Register yourinstrument ......................................................... 7
Exterior instrument parts ......................................................... 7
■CHAPTER2Installation ............................................................ 8
Operating environment ........................................................... 8
Install theinstrument ............................................................ 8
Turn ON theinstrument .......................................................... 8
Sign in to the instrument with a new Thermo Fisher™Connect Platformaccount ........ 9
Sign in to the instrument with an existing Thermo Fisher™Connect Platformaccount ... 11
■CHAPTER3Protocols ............................................................ 12
Protocols screen ............................................................... 12
Protocolfunctions .............................................................. 13
Count parameters .............................................................. 13
Load a protocol ................................................................ 14
Create a protocol .............................................................. 14
Edit a protocol ................................................................. 15
Import a protocol .............................................................. 16
Export a protocol .............................................................. 16
■CHAPTER4Slide preparation and operation ................................... 18
Recommendations ............................................................. 18
Load Countess™Cell Counting ChamberSlide ..................................... 18
Load the Countess™ReusableSlide .............................................. 20
Countess™3 Automated Cell Counter User Guide 3

■CHAPTER5Cell count and cell viabilityassays ................................ 22
Count cells inbrightfield ........................................................ 22
Rapid capture ............................................................. 22
Customize capture ......................................................... 22
Next steps ................................................................ 24
View results ................................................................... 25
Results screen forbrightfield ................................................ 25
Identify objects counted ........................................................ 25
Results screen ............................................................ 25
Identify cells counted in cell count and cell viabilityassays ...................... 25
Histogram count results ......................................................... 27
View histogram ........................................................... 27
View scatterplot ........................................................... 28
Gate count results ............................................................. 28
Gating screen ............................................................. 28
Gate count results ......................................................... 29
Save as new protocol ........................................................... 29
Edit and save a new protocol ................................................ 29
■CHAPTER6Calculators .......................................................... 30
Pre-Dilution Calculator .......................................................... 30
Cell Splitting Calculator ......................................................... 31
■CHAPTER7Save results ......................................................... 33
Save screen ................................................................... 33
Save procedure ................................................................ 33
Report ........................................................................ 36
Reportfile ................................................................ 36
■CHAPTER8Settings overview ................................................... 37
Settings screen ................................................................ 37
Connect to a wirelessnetwork ................................................... 37
Connect to a networkdrive ...................................................... 37
Software update ............................................................... 38
Guidelines for software update .............................................. 38
Update the Countess™3/3 FL Cell Counter software ........................... 38
Set the date andtime ........................................................... 39
Change your cloud region ....................................................... 42
Contents
4Countess™3 Automated Cell Counter User Guide

■CHAPTER9Maintenance ......................................................... 43
Instrument care ................................................................ 43
General guidelines for care .................................................. 43
Powersupply ............................................................. 43
Clean the cell counter .......................................................... 44
Introduction ............................................................... 44
Clean the touchscreen ..................................................... 44
Clean the instrumentcase .................................................. 44
Decontaminate theinstrument ............................................... 45
■CHAPTER10Installation/OperationalQualification ............................ 46
Generate and activate alicense .................................................. 46
Execute the Installation Qualification(IQ) .......................................... 47
Execute the Operational Qualification(OQ) ........................................ 52
■APPENDIXATroubleshooting .................................................... 57
■APPENDIXBTechnicalspecifications ........................................... 60
Physical characteristics ......................................................... 60
Technicalspecifications ......................................................... 60
Optics ........................................................................ 60
Analysisslide .................................................................. 61
Networking requirements ....................................................... 61
■APPENDIXCCountess™3 Automated Cell Counter andaccessories ....... 62
Accessory products ............................................................ 62
■APPENDIXDCSV file formatdefinition ......................................... 63
Overview ...................................................................... 63
■APPENDIXESafety ............................................................... 66
Chemicalsafety ................................................................ 67
Biological hazardsafety ......................................................... 68
■APPENDIXFDocumentation and support ...................................... 69
Customer and technical support ................................................. 69
Limited product warranty ........................................................ 69
Contents
Countess™3 Automated Cell Counter User Guide 5

Product information
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Contents
The Countess™3 Automated Cell Counter is shipped with the components listed below.
Component Quantity
Countess™3 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
Countess™3 Disposable Slide Holder 1 each
Countess™3 Reusable Slide Holder 1 each
Countess™3 USB Drive 1 each
Countess™3 Automated Cell Counter Quick Reference Card 1 each
Product description
The Countess™3 Automated Cell Counter is a fully automated cell counter and assay platform that uses
exceptional optics and image analysis algorithms to analyze trypan blue stained samples in suspension.
• The Countess™3 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 in disposable Countess™Cell Counting
Chamber Slides. Each chamber slide contains two enclosed chambers to hold the sample and
allow measurement of 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 Automated Cell Counter also provides information on cell size.
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6Countess™3 Automated Cell Counter User Guide

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8 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.
Exterior instrument parts
4
5
6
2
1
3
1Touchscreen display: The 7-inch capacitive display is the main user interface of the Countess™3 Automated Cell
Counter. It contains the buttons for all instrument functions and displays data from the cell count.
2Front 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 any 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.
3Slide port: Used to insert the analysis slide containing the sample into the counter.
4Power 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.
5Rear USB port: An additional USB port to perform functions such as plugging in a USB mouse for instrument
control.
6Power 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
Upon receiving the instrument 1
Countess™3 Automated Cell Counter User Guide 7

Installation
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.
Install the instrument
1. Unpack the instrument and place the instrument on a flat, level, dry surface.
2. Remove the thin plastic protector film from the touch-screen display.
3. Plug one end of the power cord appropriate for your region into the instrument.
4. 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39). Press Next.
•Upon completion, press Done. You will proceed to the Home screen and can begin using the
instrument as described below.
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8Countess™3 Automated Cell Counter User Guide

1. 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7.
The instrument will walk you through an initial Out-of-Box Experience to set up the instrument for
first use.
2. 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.
3. To change the current template or add a new template to the instrument, press (Templates).
Templates allows you to create customized count preferences (i.e., gate counts based on cell size,
brightness, and/or circularity). See “Load a protocol” on page14.
4. To change instrument settings, press (Settings).
Settings allows you to update the Countess™3 instrument software, customize instrument settings,
view instrument details, and export error logs. See “Settings screen” on page37.
Sign in to the instrument with a new Thermo Fisher™
Connect Platform account
Note: A Wi-Fi adapter needs to be installed and a Thermo Fisher™Connect Platform account is
required for linking. See http://thermofisher.com/connect for details on using the Connect platform.
1. Press (Sign in) from the Home screen.
2. Press Link account.
Chapter2Installation
Sign in to the instrument with a new Thermo Fisher™Connect Platform account 2
Countess™3 Automated Cell Counter User Guide 9

3. 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 Connect platform account.
4. 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.
Chapter2Installation
Sign in to the instrument with a new Thermo Fisher™Connect Platform account
2
10 Countess™3 Automated Cell Counter User Guide

Sign in to the instrument with an existing Thermo Fisher™
Connect Platform account
1. Press (Sign in) from the Home screen.
2. Select your Username from the drop-down box. Enter your PIN.
3. Press Sign in.
Chapter2Installation
Sign in to the instrument with an existing Thermo Fisher™Connect Platform account 2
Countess™3 Automated Cell Counter User Guide 11

Protocols
Protocols screen
Protocols allows you to create and save customized protocols. Each custom protocol defines the count
parameters (size, brightness, and circularity) and enables storing channels to be captured, calculator
default values, and save settings for a consistent and streamlined workflow.
• Protocols can be accessed from the Home and Gating screens.
• The currently selected protocol is displayed at the bottom of the Home screen.
• Automatic instrument functions and count parameters are defined in the Edit protocol screen. See
“Create a protocol” on page14 or “Edit a protocol” on page15.
• The Default protocol contains default count settings and cannot be edited.
• The count parameters specified in the selected protocol are applied to all new cell counts.
• Protocols can be sorted by pressing Name, Channels, or Last Modified headers.
• If you have already performed a count, loading a new protocol from the Results screen applies the
count preferences to the current count results (total cells, viability, etc.) and to all new counts.
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12 Countess™3 Automated Cell Counter User Guide

Protocol functions
The Actions screen allows you to edit, import, export, and delete protocols. Edit can be used to modify
the currently selected protocol, while Import, Export, and Delete can be used on multiple protocols.
Count parameters
Count parameters are adjusted in the Edit Protocol screen using the Gating sliders. Moving the top
and bottom gate boundaries adjusts the range of size, brightness, and circularity parameters.
4
1
2
3
1Size 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.
2Brightness 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.
3Circularity 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.
4Gating Boundary Sliders
Live or Dead can be selected only on a brightfield count. For a fluorescence count using the Countess™
3 FL instrument, select the individual channels.
•Setup – Press Setup to select the required channels (e.g., BF, GFP, TX Red) for capture. BF
(brightfield) is checked by default and cannot be deselected.
•Calculators – Press Calculators to access the Pre-Dilution and Cell Splitting Calculators and store
default calculator values. For detailed protocols, see Chapter 6, “Calculators”.
•Save – Press Save to save default save settings and your data to the cloud or a USB. For details,
see “Save screen” on page33.
Chapter3Protocols
Protocol functions 3
Countess™3 Automated Cell Counter User Guide 13

Load a protocol
1. Press (Protocols).
2. Press the desired protocol to select, then press Load.
3. To return to the previous screen without loading the new protocol, press Cancel or (Back).
The instrument will keep the saved protocol, but will return to the previous screen without loading
it.
Note: The protocol name will be displayed in the upper left corner of the count screen.
If a saved protocol has been changed and there are unsaved changes, an asterisk (*) will follow the
name.
Create a protocol
1. Press (Protocols).
2. Press Create. To assign a name to the new protocol or to change the name of the existing
protocol, press the Protocol name text box to open the keypad.
3. Type in the desired protocol name. To enter symbols, press the symbol (@%&) key. To return to the
alpha-numeric keypad, press ABC.
4. Press Enter to save the name and return to the New Protocol screen. Press to return to the
New Protocol screen without saving the name.
Chapter3Protocols
Load a protocol
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14 Countess™3 Automated Cell Counter User Guide

5. Press Save to save the new protocol.
6. Press Cancel or (Back) to return to the Protocols screen without saving.
Edit a protocol
1. Press (Protocols).
2. Select an existing protocol, then press Actions.
Note: The default protocol contains default count settings and cannot be edited.
3. Press Edit.
The Edit screen for the selected protocol opens.
4. Press the appropriate channel (BF or other dye) and edit the desired parameters to create a custom
protocol. Additionally, use the Setup, Calculators, and Save buttons to set up any of the needed
parameters to complete the custom protocol. For details, see “Count parameters” on page13.
5. Press Save.
From the Protocols screen, select the desired protocol and press Load to use that protocol to
read the sample.
Chapter3Protocols
Edit a protocol 3
Countess™3 Automated Cell Counter User Guide 15

Import a protocol
1. Press (Protocols).
2. Press Actions.
3. Press Import.
4. Press the import location Cloud or USB.
a. 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.
b. For USB, insert your USB drive into the slot on the front of the instrument.
5. Press Next.
6. Select one or more protocols from the list.
7. Press Import.
8. Once complete, press Done.
Export a protocol
1. Press (Protocols).
2. Press Actions.
3. Press Export.
Chapter3Protocols
Import a protocol
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16 Countess™3 Automated Cell Counter User Guide

4. Select one or more protocols from the list.
5. Press Next.
6. Press the export location Cloud or USB.
a. 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.
b. For USB, insert your USB drive into the slot on the front of the instrument.
7. Press Export.
8. Once complete, press Done.
Chapter3Protocols
Export a protocol 3
Countess™3 Automated Cell Counter User Guide 17

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×104to 1×107cells/mL, but the optimal range is 1×105
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
1. 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.
2. 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.
4
18 Countess™3 Automated Cell Counter User Guide

3. 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.
4. Use the touchscreen to run the sample count. See Chapter5, “Cell count and cell viability assays”.
5. 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.
6. (Optional) To remove the slide adapter, gently squeeze the tabs and pull the adapter completely out
of the instrument.
Chapter4Slide preparation and operation
Load Countess™Cell Counting Chamber Slide 4
Countess™3 Automated Cell Counter User Guide 19

Load the Countess™Reusable Slide
1. 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.
2. 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™Reusable
Slide has a 10‑µL sample capacity. Do not overfill the slide chambers.
3. Load the reusable glass slide into the reusable slide holder.
4. Insert the reusable slide holder containing the slide into the slide port and gently push into the
instrument until it clicks into place.
5. Use the touchpad to run the count.
6. 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.
Chapter4Slide preparation and operation
Load the Countess™Reusable Slide
4
20 Countess™3 Automated Cell Counter User Guide