ThermoFisher Scientific Invitrogen Countess II User manual

For Research Use Only. Not for use in diagnostic procedures.
Countess™II Automated Cell Counter
Catalog Number AMQAX1000
Publication Number MAN0014293
Revision B.0

2 Countess™II Automated Cell Counter User Guide
Information in this document is subject to change without notice.
DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC 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
MAN0010644
Revision
Date
Description
B.0 30 June 2017 Add info about edited profile indicator, save profile from results screen, dilution
calculator, and reports.
A.0 01 September 2015 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.
Manufacturer:
Life Technologies Corporation | 22025 20th Ave SE | Bothell, WA 98021
Trademarks:
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
© 2017 Thermo Fisher Scientific Inc. All rights reserved.

Countess™II Automated Cell Counter User Guide 3
Contents
About this guide............................................................................................................5
1. Product information ................................................................................................7
Product description......................................................................................................................................... 7
Instrument exterior components ................................................................................................................... 8
2. Getting started ........................................................................................................9
Installation........................................................................................................................................................ 9
Load profile .................................................................................................................................................... 11
3. Cell count and cell viability assays........................................................................15
Prepare sample .............................................................................................................................................. 15
Count cells ...................................................................................................................................................... 16
4. Results ..................................................................................................................18
View results.................................................................................................................................................... 18
Identify the cells counted ............................................................................................................................. 19
Graph count results....................................................................................................................................... 20
Gate count results.......................................................................................................................................... 21
Save as new protocol..................................................................................................................................... 22
5. Dilution calculator................................................................................................. 24
Calculate dilution .......................................................................................................................................... 24
6. Save results ..........................................................................................................27
Save count results.......................................................................................................................................... 27
Report.............................................................................................................................................................. 30
7. Instrument settings...............................................................................................31
Overview ........................................................................................................................................................ 31
Software update............................................................................................................................................. 32
Date/Time ...................................................................................................................................................... 33
8. Maintenance..........................................................................................................35
Instrument care.............................................................................................................................................. 35
Clean the Countess™II Automated Cell Counter ..................................................................................... 36
Set nominal focus .......................................................................................................................................... 37
Appendix A: Troubleshooting...................................................................................... 39
Appendix B: Product specifications ............................................................................ 41
Technical specifications ................................................................................................................................ 41
Appendix C: Ordering information.............................................................................. 42
Countess™II Automated Cell Counter ....................................................................................................... 42
Accessory products ....................................................................................................................................... 42

4 Countess™II Automated Cell Counter User Guide
Appendix D: CSV file format........................................................................................ 43
CSV file format, explained ........................................................................................................................... 43
Appendix E: Safety ......................................................................................................44
Safety conventions used in this document................................................................................................. 44
Symbols on instruments ............................................................................................................................... 45
Safety labels on instruments ........................................................................................................................ 47
General instrument safety ............................................................................................................................ 48
Chemical safety.............................................................................................................................................. 49
Chemical waste safety................................................................................................................................... 50
Electrical safety .............................................................................................................................................. 51
Biological hazard safety................................................................................................................................ 52
Safety and electromagnetic compatibility (EMC) standards ................................................................... 53
Documentation and support .......................................................................................54
Obtaining support ......................................................................................................................................... 54

Countess™II Automated Cell Counter User Guide 5
About this guide
Audience
This user guide is for laboratory staff operating, maintaining, and analyzing data
using the Countess
™
II Automated Cell Counter.
User documentation
The guides listed below are available for the Countess™II Automated Cell
Counter.
Guide Pub. no.
Countess™II Automated Cell Counters User Guide MAN0014293
Countess™II and Countess™II FL Automated Cell
Counters Quick Reference Card (QRC)
MAN0010826
Additional resources are available on the Countess™Technical Resources page.
Go to www.thermofisher.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 page 6.
Convention Use
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:
Select More Adjust.
User attention
words
Two user attention 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.

6 Countess™II Automated Cell Counter User Guide
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
defined 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 figure that contains a hazard symbol. These hazard
symbols are identical to the hazard symbols that are affixed to the instruments
(see “Safety symbols” in Appendix E).

Countess™II Automated Cell Counter User Guide 7
1. Product information
Product description
Product contents
The Countess™II Automated Cell Counter is shipped with the following
components.
Component Quantity
Countess™II Automated Cell Counter (Cat. no. AMQAX1000) 1 each
Power Cord with 4 adaptor cords
(for U.S./Canada/Taiwan/Japan, Europe, or UK)
1 each
Countess™Cell Counting Chamber Slides (50 slides/box) 1 box
Countess™II Disposable Slide Holder 1 each
Countess™II USB drive 1 each
Countess™II Automated Cell Counter Quick Reference Card 1 each
Countess
™
II
Automated Cell
Counter
The Countess™II Automated Cell Counter is a fully automated cell counter and
assay platform that uses state-of-the-art optics and image analysis algorithms to
analyze trypan blue-stained cells in suspension.
•The Countess™II Automated Cell Counter offers 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 in disposable Countess™
Cell Counting Chamber Slides (page 15). Each chamber slide contains two
enclosed chambers to hold the sample to allow you to measure two different
samples or perform replicates of the same sample.
•The Countess™II Automated Cell Counter takes 10 seconds per sample for a
typical cell count and is compatible with a wide variety of eukaryotic cells. In
addition to cell count and viability, the instrument also provides information
on cell size.
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 page 9 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™II Automated
Cell Counter.

8 Countess™II Automated Cell Counter User Guide
Instrument exterior components
Touch-screen display:The 7-inch capacitive touch-screen display is the main user interface of the
Countess™II Automated Cell Counter. It contains the buttons for all instrument functions and displays
data from the cell count.
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.
Slide port: The slide port is used to insert the Countess™Cell Counting Chamber Slide containing the
sample into the instrument.
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.
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.
Note: The Countess™II Automated Cell Counter (Cat. no. AMQAX1000) uses brightfield
illumination only and does not support the EVOS™LED light cubes.

Countess™II Automated Cell Counter User Guide 9
2. Getting started
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 affect the image quality.
•Operating temperature range: 4°–40°C (39°–104°F)
•
Relative humidity range: <80% (non-condensing)
IMPORTANT! Do not position the instrument so that it is difficult to turn off
the main power switch located on the back of the instrument (see page 8). 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.

10 Countess™II Automated Cell Counter User Guide
Turn ON the
instrument
1.
Turn on the instrument by flipping the power switch on the back of the
instrument (page 8) to the ON position.
The instrument initializes and displays the Home screen.
2. From the Home screen, you can proceed immediately to the assays by inserting
a slide (page 15).
Alternatively, you can change or add a profile (Step 3, below) or change
instrument settings (Step 4, below).
3. To change the current profile or to add a new profile to the instrument,
press the Profilesbutton in the upper left corner.
Profiles allow you to create customized count preferences (i.e., gate counts
based on cell size, brightness, and/or circularity) (page 11).
4. To change instruments settings, press the Instrument Settings button in
the upper right corner.
Instrument settings allow you to update the Countess™II software and change
the date and time (page 31).

Countess™II Automated Cell Counter User Guide 11
Load profile
Profiles screen
Profiles screen allows you to create and save up to 9 customized profiles. Each
custom profile defines the count parameters (size, brightness, and circularity) and
the Auto Lighting function for a consistent and streamlined workflow.
•You can access the Profiles screen from the Home, Capture, Results, Advanced,
or Adjust screens.
•The current profile is displayed on the upper left corner of
the Home, Capture, Results, Advanced, or Adjust screens.
•The Auto Lighting function and count parameters are set in the Edit profile
screen (page 12).
•The Default profile contains default count settings and cannot be edited.
•The count parameters specified in the selected profile are applied to all new
cell counts.
•If you have already performed a count, loading a new profile 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 setting that is saved as part of the
protocol (size, brightness, or circularity) on the Results
screen, the profile name is appended with the (*) symbol.

12 Countess™II Automated Cell Counter User Guide
Edit profile screen
The Auto Lighting function and count parameters are set in the Edit profile screen.
Auto Lighting
Auto Lighting function automatically illuminates the sample in the brightfield
channel for increased sample-to-sample consistency and decreased user-to-user
variability. By default, the Auto Lighting function is turned on; to toggle it on and
off, use the Auto Lighting checkbox (see page 13).
Count parameters
Count parameters are set using the parameter sliders, which correspond to a single
channel that is selected via the channel selection radio buttons.
•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.
= larger objects = smaller objects
•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.
= brighter objects = dimmer objects
•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.
= less circular = more circular
•Size and brightness 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 Automated Cell Counter User Guide 13
Load a profile
1.
Press the Profiles button located on the upper left corner of the screen to
open the Profiles screen.
2. Press the desired profile to select, and then press Load.
The instrument will load the count parameters specified in the selected profile
and return to the previous screen.
3. To return to the previous screen without loading the new profile, press
the previous button.
The instrument will keep the saved profile, but return to the previous screen
without loading it.
Add/edit a profile
1.
Press the Profiles button located on the upper left corner of the screen to
navigate to the Profiles screen.
2. To add or edit a new profile, select an empty or an existing profile, and then
press Edit. The Edit screen for the selected profile opens.
Note: The Default profile contains default count settings and cannot be edited.
3. Select or deselect the Auto Lighting checkbox to turn the Auto Lighting
function ON or OFF.
4. To define the new count parameters, select Live or Dead from the Trypan Blue
radio buttons.
5. Adjust the size, brightness, and circularity thresholds using the appropriate
parameter slider (page 12).
Note: Although the Auto FL Threshold checkbox and the FL channel
radio buttons are visible on the Edit profile screen, they are not
functional on Countess™II Automated Cell Counters, which only
feature a single brightfield channel.

14 Countess™II Automated Cell Counter User Guide
6.
To assign a name to the new profile or to change the name of the existing
profile, press the Profile name text box. The alpha-numeric keypad opens.
7. Type in the desired profile name using the alpha-numeric keypad.
To enter symbols, press the symbol (@%&) key. To return to the alpha-
numeric keypad, press ABC.
8. Press Enter to save the name and return to the Edit profile screen.
To return to the Edit profile screen without saving the name, press the
close button.
9. Press Save to save the new profile, and then press Close in the confirmation
screen to return to the Profiles screen.
To return to the Profiles screen without saving, press Cancel.
10. On the Profiles screen, press Load. The instrument will load the count
parameters specified in the selected profile and return to the previous screen.
11. To return to the previous screen without loading the new profile, press
the previous button. The instrument will keep the saved profile, but
return to the previous screen without loading it.

Countess™II Automated Cell Counter User Guide 15
3. Cell count and cell viability assays
Prepare sample
Recommendations
To obtain the best results, follow these recommendations:
•Ensure that the cell sample is homogeneously mixed.
•The measurement range extends from 1 × 104–1 × 107cells/mL, but the optimal
range is 1 × 105–4 × 106cells/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
™
Chamber Slide
1.
Prepare the sample by adding 10 μL of your cell suspension to 10 μL of 0.4%
trypan blue stain. Mix the sample mixture well by pipetting up and down a
few times.
2. Gently pipet 10 µL of the trypan blue-stained sample into the half moon-
shaped
sample loading area. The sample is loaded into the chamber through capillary
action.
Note: Each chamber in the Countess™Cell Counting Chamber Slide has a
10-µL sample capacity. Do not overfill the slide chambers.
3. Let the sample mixture settle in the chamber for 30 seconds, and then insert the
slide into the slide port (see page 8). You will hear a soft click, if the slide is
pushed in correctly.
4. To remove the slide, 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.
Note: After using the Countess™Cell Counting Chamber Slides,
appropriately dispose of them as biohazardous waste. Do not reuse the
disposable chamber slides.

16 Countess™II Automated Cell Counter User Guide
Count cells
Count procedure
1.
Load the Countess™Cell Counting Chamber Slide with your trypan blue-
stained sample and insert it into the slide port of the instrument as described
on page 15. You will hear a soft click, if the slide is pushed in correctly.
2. When the slide is inserted, the instrument automatically illuminates the sample,
sets the intensity of brightfield illumination, and auto focuses on the cells.
Note: To turn off the Auto Lighting function, see page 12.
3. Optional: Press the Profiles button and load the desired profile
as described on page 13.
4. Optional: If desired, press the Focus button, and then use the Focus slider
to manually bring your sample into focus (see Note below).
If needed, Zoom in on the image while adjusting the focus.
Note: The auto focus algorithm is designed to highlight the differences
between live and dead cells. Therefore, the optimal focus level is where
the “live” cells have a light colored center and the “dead” cells are dark
throughout. To enable optimal auto focus functionality, you may need
to initially refine the focus by adjusting it manually and then setting it
as the nominal focus. For more information, see page 37.
5.
Optional: Manually adjust exposure using the light source slider.
The light source slider controls the LED intensity, camera gain, and exposure
time and it is used for adjusting the image brightness.

Countess™II Automated Cell Counter User Guide 17
6.
Press Capture. The instrument temporarily captures the image and displays
the results (total concentration, percentage and concentration of live and dead
cells). For more information, see “Results”, page 18.
Next steps
•
To identify the objects (i.e., cells) counted as “live” or “dead”, press More to
navigate to the Advanced screen (page 19).
•To see the distribution of live and dead cells in a graphical format, press the
Graph button (page 20).
•To gate the results by object size, brightness, or circularity, first press More to
open the Advanced screen, and then press Adjust to navigate to the Adjust
screen (page 21).
Note: You can save the changes you make to the size, brightness, or circularity
parameters in the Adjust screen to the current profile or as a separate profile
directly from the Advanced screen (see “Save as new protocol”, page 22).
•To calculate the volume of cell sample and buffer needed to reach a desired
concentration based on the count results, press Dilution Calculator to open the
Dilution Calculator application (page 24).
•To permanently save the results, press Save (page 27).
•To perform a new count, remove the slide and reinsert it into the instrument to
count the sample in the second chamber.

18 Countess™II Automated Cell Counter User Guide
4. Results
View results
Results screen
The Results screen for cell count and cell viability assays displays a composite
image of the objects counted and the results of the cell count and cell viability
calculations (total concentration, percentage and concentration of live and dead
cells).
Note: When performing brightfield (BF) cell counts and cell viability assays,
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 Automated Cell Counter User Guide 19
Identify the cells counted
Advanced screen
The Advanced screen allows you to identify the objects (i.e., cells) counted in each
channel and included in the count results for further review. After reviewing the
marked objects, you can adjust the threshold for size, brightness, and/or circularity
as desired for your application (see “Gate count results”, page 21).
Identify Live and
Dead cells
1. On the Results screen, click More.The Advanced screen opens.
2. To identify the cells that are included in the count as “live”, press the Live
button. “Live” cells will be circled in green on the screen.
To identify the cells that are included in the count as “dead”, press the Dead
button. “Dead” cells will be circled in red on the screen.
Note: You may select either or both options. In the example below, both Live
and Dead buttons are pressed and “live” and “dead” cells are marked with
green and red circles, respectively.
3. To unmark the cells identified as “live” (green) or “dead” (red) on the screen,
press the Live or the Dead button again, respectively.

20 Countess™II Automated Cell Counter User Guide
Graph count results
View graph
For cell count and cell viability assays, you can view the distribution of cells (live
and/or dead) based on size in a graphical format.
Note: You can view the Graph on Results, Advanced, and Adjust screens.
1. To view the graph showing the distribution live and/or dead cells based
on cell size, press the Graph button.
2. To view the distribution of only the live or dead cells, check the corresponding
Live or Dead check box on the graph.
The graph will automatically update and display the distribution of cells based
on size only in the selected population.
3. Optional: Using the size, brightness, and circularity sliders, adjust the count
parameters. As you adjust the count parameters, the count results and the
graph will be automatically updated.
4. To close the graph, press the Graph button again.