Countess™ II Automated Cell Counter - FAQs

Two things may be happening. Manual hemocytometer counting can be fraught with error, due to user bias and calculation errors. If the cells are clumping, however, the Countess counter may count the clump as one cell (although usually it can discriminate). Look at the images from the Countess counter and the circles it draws over counted cells to see if this is the issue. If so, you can increase the sensitivity of the instrument by adjusting the parameters.

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There are a couple of possibilities:

First, check to see if the settings have been changed (size, circularity and brightness). Another user might have changed the parameters.

Second, make sure your cells are within the size range for the assay (4–60 µm).

Finally, carefully focus the samples.

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What you see on the screen is what is counted. Regions that are not visible are not counted. So if the cells are circled correctly, the cell count should be correct.

Open up the histogram window when setting gates, so that you can see what is included and excluded from the count. It is also a good idea to check the histogram just to make sure any gates have not been set incorrectly even if you are not adjusting them. Be sure to set the same gates for both live and dead cells. You can save gates as a profile so that they are applied the same way to all the samples. Brightfield viability is only going to be accurate for cells with diameters >7 µm, but the instrument will see cells as small as 1 µm. So, if you have a lot of cells with diameters lesser than ~5 µm, we recommend setting a gate on both live and dead cells to remove these small cells from the count and improve the live percentage. Otherwise, these small cells will be counted as dead regardless of whether they are dead or live.

Note: If you do see a lot of 1-2 µm counts (seen in the histogram), it could be due to precipitates in the trypan blue stain, which is common. The three approaches below may help minimize trypan blue precipitate in your cell counting samples: - Sterile filtration of trypan blue using 0.3 micron filter
- Centrifugation or passive settling of trypan blue solution (avoid mixing or vortexing trypan stock solutions)
- Gentle warming of the 1 mL trypan blue solution to 37 degrees C for 10 mins

The following are parameters to keep in mind to ensure accurate viability determination:

- The Countess II must be able to find one pixel in the center of the cell that is brighter than the edge pixels to be counted as a live cell. So focusing through the widest diameter of the cell for small cells is important.
- Contrast just needs to be a shade of grey, not super black or white for the Countess II to see the cells well.
- Let the cells settle for about 20 seconds before counting (especially for small cells), so that they are all in the same focal plane on the bottom of the slide.
- Be sure that your total cells are less than 1 X 10E7 cells/mL. A good concentration range is about 100-300 cells visible on the screen. If you want, you can find actual numbers of cells counted in the .csv file. If cells are too confluent or if a lot of small debris is present, the Countess II might have a hard time figuring out what is background and be unable find the cells correctly. Clumping is normally not a problem as long as all the cells are in the same focal plane. If no gates are applied and cells are still not circled correctly, we recommend diluting the sample.

To set the nominal focus, please complete the following steps:
- Put in a slide with cells stained with Trypan Blue stain.
- Click the focus icon at the bottom of the count screen; you may also zoom in if desired.
- Use the focus slider to find a good focus. A good focus would be one where your live cells are showing bright centers with defined edges and your dead cells are showing totally dark.
- Once you have a good focus, tap the "set" box on the top of the focus slider.
- Reinsert the slide and allow the autofocus to work from the new nominal focus point. If the nominal focus is not set near a good focus for your cells, the autofocus may be looking in the wrong range and will not work correctly.
- Press "count".

Yes, the instrument multiplies the count by 2 to correct for the 1:1 trypan blue dilution.

Unfortunately, the Countess II Automated Cell Counter does not work well with yeast cells. This instrument can detect cells as small as 4 µm but can only accurately determine viability in brightfield for cells 7 µm and larger. Yeast cells are just below the cut-off for determining viability.

We have validated CellEvent Caspase 3/7 Green Detection Reagent (Cat. No. C10423) in combination with SYTOX Red Dead Cell Stain (Cat. No. S34859) for discriminating apoptotic cells from dead cells using the Countess II FL Automated Cell Counter. For further details, please see this Application Note (http://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Laboratory%20Instruments/Files/0615/CO210384-CountessIIFL-Apoptosis-AppNote.pdf).

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We have validated the following kits for use on the Countess II FL Automated Cell Counter:

LIVE/DEAD Viabilty/Cytoxicity Kit (Cat. No. L3224) containing calcein AM and ethidium homodimer-1
ReadyProbes Cell Viability Imaging Kit, Blue/Green (Cat. No. R37609)
ReadyProbes Cell Viability Imaging Kit, Blue/Red (Cat. No. R37610) containing NucBlue Live/NucGreen Dead and NucBlue Live/propidium iodide
See this Application Note for details - https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Laboratory%20Instruments/Files/0415/CO014723-Countess-II-FL-Viability-Appnote_FHR.pdf.

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Yes, the Countess II FL instrument uses the same light cubes as the EVOS imaging systems. The Countess II instrument does not use light cubes.

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The Countess II or Countess II FL instrument can also be controlled by a USB mouse, so you can try reinstalling the software by plugging a USB mouse into either USB port on the instrument and use the mouse to perform the software update.

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The drive must be FAT32 formatted; verify this before proceeding. At present, you should assume that all other formats will not work.
The update file must sit on the top level of the USB drive, not within a folder or subfolder.
File cannot be renamed in any way.
File cannot be zipped or compressed during distribution. It must be uncorrupted during transfer.
The device takes a few seconds to recognize the USB drive.
If needed, check that the USB port is functional by testing a USB mouse.
To format a USB to FAT32:

Insert drive requiring reformatting into a PC, find dive in “My computer”
Right click on USB drive, select Format.
Select file system, FAT32.
Click Start.
Reformatting will result in the loss of all files from the USB drive, so ensure that you have copies of any important files on another drive prior to reformatting. After reformatting, the software should be retransferred onto the USB drive prior to updating the Countess II or Countess II FL software.

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Ensure that the cells are focused correctly so that live cells have bright centers and dead cells are dark throughout. If the cells are not well focused and look dark on the screen, the Countess II or Countess II FL instrument will count them as dead cells. If cells are well focused, have bright centers, and are being counted as dead, confirm that they are within the appropriate cell size range and try adjusting the settings. If cells are exposed to trypan blue for a long period of time, viability could be affected so the slide should be prepared and counted fresh each time for best results.

In some cases, heavily pigmented cells will also look dead in brightfield; such cells may require fluorescent viability stains.

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There are two predominant reasons for this: either the cells in question are truly dead or the focus should be adjusted. In some cases, heavily pigmented cells will also look dead in brightfield; such cells may require fluorescent viability stains.

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If you are pipetting different samples from the same cell sample, the variability could be due to pipetting or mixing. Use recently calibrated pipettors and make sure that the cell suspension is well mixed by pipetting up and down several times before you add 10 µL to trypan blue. Once the cell sample is mixed with trypan blue, it should also be well mixed by pipetting up and down and loaded into the Countess slide right away.

If you are counting replicates of the exact same slide, visually inspect that all cells are counted correctly in the image. Ensure that you do not shake or agitate the slide between counts. There may be a slightly different field of view each time a slide is inserted, so depending on the concentration and uniformity of the cells, there will be some variability when performing replicate counts of the same slide, although it should be less than 10%. When counting fewer cells, a small field of view change for only a small number of cells can have a larger affect, so variability can be reduced by counting cells at a higher concentration.

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Ensure that a FAT-formatted USB drive is inserted in the instrument.

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The brightfield cell concentration assumes that you have diluted the cells 1:1 in trypan blue, so it already takes this dilution into account and multiples the values by 2 so that the cell concentration displayed is the original cell concentration before dilution in trypan blue. The total cell concentration displayed after a fluorescent count does not take any dilution into account, so it is the actual cell concentration in the slide.

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The light source adjustment controls the LED intensity, camera gain, and exposure time, and it is used for adjusting the image brightness and contrast before you capture the image. Once the cells are inserted, there will be a bar on the right hand side to adjust the light source intensity. Often it helps to turn it down so that the background is slightly darker, which allows better contrast between live and dead cells. Optimal light intensity in brightfield is usually around 10-20 if there are no light cubes in the instrument, and slightly higher around 20-30 if there are light cubes present, but this could vary depending on the sample.

Once the image is captured, you can also adjust the range of cell size and cell brightness that the counting algorithm will count. This doesn't change the actual brightness of the image or how it looks, but determines which cells will be counted. Once you press “Capture” to count the cells, you can hit the “Adjust” button and make sure that the size, brightness, and circularity gates are all maximized by maximizing the slider bars to include all of the cells in the count. When you go into the “Adjust” screen, you can adjust the size, brightness, and circularity for both live and dead cells, so make sure that you select both the live and dead buttons in the adjust screen so that you are completely maximizing the size and brightness gates for both the live and dead cell population. This is mainly an issue if there are cells that appear in the image, but aren't being counted. It's best to expand the gates first, make sure all of the cells are counted, and then narrow them if you want to exclude cells of a certain size or certain brightness.

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The screen has a thin plastic film screen protector that should be removed before use.

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Once you press “Capture” to count the cells, hit the “Adjust” button and make sure that the size, brightness, and circularity gates are all maximized to include all of the cells in the count. When you go into the “Adjust” screen, you can adjust the size, brightness, and circularity for both live and dead cells, so make sure that you select both the live and dead buttons in the adjust screen so that you are completely maximizing the size and brightness gates for both the live and dead cell population. It's best to expand the gates first, make sure all of the cells are counted, and then narrow them if you want to exclude cells of a certain size or certain brightness.

You can also select the “Default” user profile to restore the size, brightness, and circularity gates to their maximum. If the gates are fully maximized, the CSV should indicate 0-60 for cell size min and max and 0-255 for brightness min and max for both live and dead cells.

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Decrease the brightfield and fluorescence light intensity before counting the cells.

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First, make sure you have the most recent version of the Countess II or Countess II FL software installed on the instrument (it is available here). You may need to manually adjust the focus and set the nominal focus so that the autofocus will have a set point to focus on the cells. Once cells are stained 1:1 in 0.4% trypan blue and placed in the slide, you may want to let the cells settle for around 30 seconds. Then place the slide in the instrument and after you let the machine autofocus, you may need to refine the brightfield focus with the manual focus. Ideally the live cells should be slightly under focused, where the centers are bright compared to the dead cells that are dark throughout. You may want to zoom in so that you can better see the cells, then manually adjust the focus using the focus slider bar before you hit capture. Once the cells are focused, you can tap the blue “set” box to set the nominal focus; setting the nominal focus will improve autofocus consistency with future slides.
Make sure there are no bubbles or debris visible on the screen that could interfere with the autofocus and make it more difficult to get the sample in the correct focal plane. You can find some more guidelines for focusing starting on page 20 of the manual.

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Uneven illumination on the screen may be fixed by resetting the light cube tray. First, make sure you have the most recent version of the Countess II or Countess II FL software installed on the instrument (it is available here). If the software is updated, the light cube tray should readjust each time the instrument is power cycled. You can also select the “change light cubes” option under the settings menu, allow the light cubes to reset, and then turn the instrument off and back on. This process may help with uneven screen illumination even if you do not have light cubes present or if you have the Countess II instrument (without the fluorescence option).

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Select the “Default” profile to restore the size, brightness, and circularity gates to their maximum.

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The brightness and size sliders establish the thresholds for the imaging algorithm, allowing users to set both the brightness and size ranges for the sample. If the parameters are at their maximum settings, all brightness levels and sizes are counted, so they cannot be further adjusted. To narrow the brightness or size range for the count, you can move the ends of the slider bar or you can hit the icons at the end of the slider bar to adjust them. To demonstrate this while on the ‘Adjust' screen, repeatedly tap the ‘dim' icon on the brightness slider or manually move the bottom end of the slider and watch the green brightness bar decrease in height. The resulting narrowed brightness range can be slid across the brightness scale to include only dim cells, only bright cells, or something in between depending on what is to be included in the count. The same adjustments can be performed for cell size.

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The most recent version of the Countess II or Countess II FL software is available here (https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/countess-software.html). There will be no more software updates to Countess II/IIFL.

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Unfortunately, there is no definitive answer to this question. The rate at which the cell-impermeant dye is absorbed depends on the cell type, their state of health, nourishment, engulfment activity, etc.

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Exposure to light may degrade the dye and these contaminants may promote precipitation. Trypan blue can also form orange/red fibrous aggregates if exposed to refrigeration or freezing temperatures.

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Trypan blue is necessary for live/dead discrimination and it provides contrast for the cells, however if viability information is not required, some cells may have enough contrast on their own to be counted in brightfield without trypan blue. The cell concentration value obtained in brightfield assumes the cells are diluted 1:1 in trypan blue, so if the cells are not diluted, you will need to divide the concentration by 2 to get an accurate concentration for cells that are not mixed with trypan blue.

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No, the Countess II and Countess II FL instruments are provided with a box of slides, but are not provided with trypan clue stain so it will need to be purchased separately (Cat. No. T10282). If you purchase a box of slides separately, the slides will come with trypan blue stain.

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Erythrosine B (0.04%) can be used a a substitute for Trypan Blue in the Countess II and Countess II FL instruments.

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The Countess II and Countess II FL instruments were optimized for mammalian cell counting; however, counting other cell types may be possible.

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We have tested over 20 commonly used cell types, including primary cells, PBMCs, insect cells, and fish cells, using the default settings. Specific cell types could require some parameter setting adjustments, and those may be optimized by the user.

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If there are two cells attached to each other with enough circular definition for each, the image analysis firmware will distinguish them as two different objects.

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The Countess II and Countess II FL instruments do not count live sperm cells or other fast-moving cells such as protozoa.

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Yes, the Countess II and Countess II FL instruments can count RBCs. However, we recommend diluting the blood sample approximately 1:10,000. The instrument cannot assess the viability of RBCs due to their pigmentation and trypan blue staining pattern.

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Yes, the Countess II and Countess II FL instruments can count PBMCs. However, they cannot differentiate between white blood cell types.

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The advanced counting algorithms of the Countess II and Countess II FL instruments can clearly identify cell boundaries within clumps of cells, typically resulting in accurate cell counts.

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Yes, but you may need to experiment with several circularity settings until you find the one that is best for your cell type.

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No, bacteria are too small to be distinguished from non-cell debris.

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Yes, the instrument can count in both fluorescence and brightfield modes, allowing simple calculation of transfection efficiency that is shown on the results screen as “% positive” for the light cube of interest.

Clean the surface of the instrument with a damp cloth. To clean the LCD screen, turn off the instrument, disconnect the power cable, and clean the LCD screen with a soft cloth lightly moistened with LCD cleansing detergent. Cleaning the screen with excessive force can damage the LCD screen. Wipe the screen dry immediately. The Countess II and Countess II FL instruments have no moving parts to maintain, no tubes to clean, and no buffers to replace.

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The Countess II and Countess II FL instruments have a very small footprint (23 cm (w) x 14 cm (d) x 23 cm (h)) and weigh about 3.6 kg (without light cubes installed). Light cubes are not included with the Countess II FL instrument; they must be purchased separately.

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The USB ports are identical, however if a USB or external drive is connected to both ports at the same time, only the first storage device connected will be recognized. A USB mouse can also be used in either of the USB ports.

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Countess II and Countess II FL instruments have no internal memory, so there are no files that need to be erased. Files can be saved to the USB as standard image files (JPEG, PNG, TIFF, or BMP) to be opened by any external imaging program, or data can be saved to the USB in a CSV file to be opened by a spreadsheet program.

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No. You can use a USB drive to transfer your data from the instrument to a computer that uses a Windows operating system.

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There is no computer software available since the data does not require any special software to open. Files can be saved to the USB as standard image files (JPEG, PNG, TIFF, or BMP) to be opened by any external imaging program, or data can be saved to the USB in a CSV file to be opened by a spreadsheet program. The software for the instruments may be downloaded here (https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/countess-software.html).

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No, the Countess II and Countess II FL instruments do not require a computer.

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Yes, the Countess II FL instrument does not require light cubes and can be used without them if fluorescence is not required.

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Yes, the Countess II FL instrument uses the same light cubes as the EVOS imaging systems. The Countess II instrument does not use light cubes. The Countess II FL instrument without light cubes installed will function exactly like the Countess II instrument-as a brightfield cell counter.

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Both the Countess II and Countess II FL Automated Cell Counters include autofocus, a touch screen, brightfield counting, and viability testing using trypan blue, and use disposable slides. The Countess II FL Automated Cell Counter can also accommodate a reusable glass slide and fluorescence detection using EVOS LED-based light cubes.

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It depends on the type of fluorescent stain used on the cells. Trypan blue is a cell-impermeant chromophore that can quench fluorescence. It may quench fluorescent staining on the surface of live cells or internal fluorescent staining in dead cells.

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The Countess II and Countess II FL instruments come precalibrated. You do not have to calibrate the instruments.

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It is based on dye exclusion using 0.2% Trypan Blue dye. Trypan Blue is excluded by the live cells and enters the dead cells; this results in intense staining of the intracellular proteins.

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The following cell lines and primary cells have been tested:

- Cell lines: HEK 293, A431, CHO-M1, CHSE, COLO-205, COS-7, HeLa, HepG2, HL-60, J774(MMM), Jurkat, K-562, MCF-7, MRC-5, NIH/3T3, PC-12, SF-21, U266, and U2OS

- Primary cells: Human adipose tissue-derived stem cells, HASMCs, HPAECs, HPASMCs, HUVECs, C2C12, RBCs, and PMBCs

We are continually testing additional cell types.

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When an experienced worker manually counts cells using a glass hemocytometer together with a microscope, count-to-count variability of a single sample is commonly 10% or more. Users of Countess II and Countess II FL instruments should typically see <5% count-to-count variability.

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When counting in brightfield mode, the Countess II and Countess II FL instruments automatically assume a 1:1 dilution has been made with trypan blue. The same assumption is not made when in fluorescence mode.

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The Countess II instruments are designed to read samples with concentrations in the range of 1 x 10E4 cells/mL to 1 x 10E7 cells/mL.

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The time taken is ˜10 seconds per sample.

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The Countess II and Countess II FL instruments can detect particles/cells ranging from 4 - 60 µm. Cells ranging from 7 - 60 µm can be counted and assessed for viability with brightfield viewing.

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