Cell Counting Support—Getting Started

We recommend adding 0.1 mL of trypan blue stock solution to 1 mL of cells (final concentration 0.04% w/v). Final concentrations may range from 0.0.04% to 0.2% (w/v) dependent upon the sample and instrumentation. 

Yes, you can find the protocol at the following link.

Trypan blue will stain cells that have a compromised membrane. It cannot differentiate between compromised membranes caused by apoptosis or necrosis.

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.

Trypan blue will bind to serum proteins as well as cellular proteins, which may result a high level of background staining. If the background is too dark, cells should be pelleted and resuspended in protein-free medium, buffer, or normal saline prior to counting.

Trypan blue is a cell impermeant stain used to estimate the number of dead cells in a viable population. Its utility is based on the fact that it is a charged dye and does not enter cells unless the membrane is compromised. Live (viable) cells exclude the dye but dead (non-viable) cells or cells with a compromised membrane are stained an intense blue.

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.

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

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

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.

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

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.

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.

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.

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.

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

No. The Countess™ II instrument does not have the electrical components to accept light cubes for fluorescence-based detection. It can only be used for brightfield detection. 

Yes, the slides have different thicknesses, so the nominal focus must be set for both the reusable and disposable slides. Once set, the Countess™ II FL instrument can detect which slide is being used and apply the appropriate nominal focus value.

A field of view, 2.2 mm x 1.6 mm is counted, which yields a ~3.5 square mm total. The Countess™ II and Countess™ II FL instruments only count a single field of view which corresponds directly to the field of view visible on the screen with no zoom applied.

Currently, we only supply the disposable slides individually wrapped.

No. The imaging software on the Countess^™ II Countess™ II FL instruments cannot detect motile cells. The cells must be immobilized or fixed prior to analysis. 

1 x 10E5 to 4 x 10E6 cells/mL is the core confidence range that will yield the best accuracy and precision. Counting samples from 1 x 10E4 to 1 x 10E7 cells/mL has been performed, but with greater count-to-count variability.

The Countess™ II and Countess™ II FL instrumentscan detect particles/cells ranging from 4–60 μm. Accurate viability assessment is possible with cells ranging from 7–60 μm.

A sophisticated image analysis algorithm identifies objects and classifies particles or cells by roundness and size, and then distinguishes live cells from dead cells by their staining pattern.

It is based on dye exclusion using 0.2% trypan blue dye. Live cells exclude the dye. Dead cells show intense staining of the intracellular proteins. 

No, unfortunately the cell by cell size and fluorescent data used to prepare the histogram cannot be accessed.

Yes, the IQ⁄OQ (Installation Qualification⁄Operational Qualification) User-Performed Test Procedures package can be purchased separately through your sales rep (Cat. No. A28154). If you require an engineer to perform the IQ⁄OQ protocol, the Cat. No. is 4479677 for a one time test, or 4479671 for reiterative test procedures.

We do not sell assays specific for the Countess™ II FL instrument, however we have validated some of our existing fluorescent viability and apoptosis assays for the Countess™ II FL instrument. Application notes for fluorescent viability and apoptosis assays can be found here.

Yes, the brightfield viability count and concentration takes the dilution into account, so the values displayed are for the original cell sample before dilution into trypan blue.

No. The trypan blue sample preparation was simplified to 1:1 mix, load, read, and the result given only takes that 2-fold dilution into account. Any further dilutions for a final answer need to be calculated by the user.

The time taken is ~10 seconds per sample.

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

When we tested the Countess™ II and Countess™ II FL Automated Cell Counters side by side with the original Countess™ Automated Cell Counter using the same cells and slides, the count-to-count variability between instruments was <10%. 

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.

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 observe <5% count-to-count variability.

No, in order to obtain brightfield viability and fluorescence information, two separate counts have to be performed.

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.

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

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

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.

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

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.

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

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.

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.

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

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

The Countess™ II FL instrument is able to work with dyes that you may already be using to measure viability. Some popular dyes that have been validated on the Countess™ II FL instrument are included in the table below:

The Countess™ II FL instrument is able to work with dyes that you may already be using to measure apoptosis. Some popular dyes that have been validated on the Countess™ II FL instrument are included in the table below:

It depends on the type of fluorescent stain used on the cells. Trypan clue is a cell impermeant, colored dye that can quench fluorescence, so it may quench fluorescent staining on the surface of live cells, or internal fluorescent staining in dead cells.

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.

As fluorescence is measured on a relative scale, this is too environmentally dependent to offer a quantitative answer. In short, the Countess™ II FL instrument offers fluorescence sensitivity similar to that of a low-magnification epifluorescence microscope.

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.

The original Countess™ Automated Cell Counterhas been discontinued. However, we still offer consumables for the instrument. The most current software and firmware for the original Countess™ Automated Cell Counterare still available here at the bottom of the page under “Original Countess Software Download”. The software is also provided on the USB drive that comes free with the instrument. This software allows you to manipulate your cell-counting data and to save your data in a pdf report format for printing or archiving.

Note: As an alternative to the original Countess™ Automated Cell Counter, we offer the Countess™ II and Countess™ II FL Automated Cell Counters.

Yes, the viability count and concentration takes the dilution into account, so the values displayed are for the original cell sample before dilution into trypan blue.

No. The trypan blue sample preparation was simplified to 1:1 mix, load, read, and the result given only takes that 2-fold dilution into account. Any further dilutions for a final answer need to be calculated by the user.

It takes 30 seconds per sample. 

The Countess™ Automated Cell Counter is designed to read samples from 1 × 10E4 cells/mL to 1 × 10E7 cells/mL, with the highest accuracy between 1 × 10E5 cells/mL and 4 × 10E6 cells/mL

The Countess™ Automated Cell Counter can count cells ranging from 5– 60 μm.

It is based on dye exclusion using 0.2% trypan blue dye. Live cells exclude the dye. Dead cells take it up into their cytoplasm.

A sophisticated image analysis algorithm identifies objects, classifies cells by roundness and size, and then distinguishes live cells from dead cells by their staining pattern.

Yes. It measures the size of live and dead cells as well as the average size of each cell population.

The original Countess™ Automated Cell Counter has a very small footprint (27 cm (w) x 20 cm (d) x 19 cm (h)) and weighs about 5.7 kg.

Currently, we only supply the slides individually wrapped.

The Countess™ instrument does not need regular maintenance service. Clean the surface of the Countess™ 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. Do not reuse the counting chambers. To troubleshoot problems with the instrument, contact Tech Support at techsupport@thermofisher.com. To avoid damaging the instrument, do not perform any repairs or service on the Countess™ instrument.

No, it does not require a computer.

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

For maximum analysis capabilities and to print a report, you can load Countess™ software on your computer. The most current software and firmware for the original Countess™ Automated Cell Counterare available here at the bottom of the page under “Original Countess Software Download”. The software is also provided on the USB drive that comes free with the instrument. This software allows you to manipulate your cell counting data and to save your data as a PDF for printing or archiving.

Yes. All of the data is saved as a .dbt file that can be opened with the Countess™ PC software. This also includes a .jpg file of the image. You can open the .jpg file with any imaging software.

Yes. You will need to use the Countess™ software on your PC to generate the report. The report shows an image of your cells, all numerical data, and a cell size histogram.

You can open the .csv file with any spreadsheet software. You can also open the image file with any imaging software. To see the complete data set, manipulate the data and print a report, you may use the Countess™ software available here at the bottom of the page under “Original Countess Software Download”. The software is also provided on the USB drive that comes free with the instrument. 

The following cell lines have been tested: 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. The following primary cells have been tested: human adipose-tissue derived stem cells, HASMC, HPAEC, HPASMC, HUVEC, C2C12 , RBC, and PMBC. 

We have not tested very irregular or elongated cells. If the cells are very irregular or elongated, try the “Parameters” function under the “Settings” menu to vary the circularity. This function alters the way that the image analysis software recognizes cells. You may need to experiment with several circularity settings until you find the one that is optimal for your cell type.

We have successfully counted Saccharomyces cerevisiae (Fleishman’s baker’s yeast), a consumer product. The Countess™ instrument successfully counted these yeast cells, as long as they were not too clumpy. However, it cannot distinguish live yeast cells from dead yeast cells.

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

The Countess™ instrument can accurately count clumps of up to about five or six cells. Above this number, cells will overlap and the instrument seems to be about as accurate as the human eye. However, the cells will be counted as larger than real size.

The Countess™ instrument can count white blood cells from lysed whole blood and Ficoll™ medium cell preparations. 

Yes. You should dilute the blood sample approximately 1:10,000 and count in “bead” mode. The instrument cannot assess the viability of cells in a whole blood sample.

Yes. The Countess™ instrument can count PBMCs. However, it cannot differentiate between different white blood cell types.

Yes. The Countess™ instrument can count RBCs. Dilute the blood sample approximately 1:10,000 and count in “bead” mode. The instrument cannot assess the viability of red blood cells.

We have not tested plant cells. If the plant cells fall within the optimal size range for the instrument and are not extremely clumpy, the Countess™ instrument should be able to count plant cells.

It is difficult to count cells that are moving quickly. The instrument does not count live sperm and fast-moving protozoa.

The instrument is able to calculate total concentration, but cannot provide viability information on the cells. Set the instrument to “bead” mode, then you can perform the cell counting.

No. The instrument cannot selectively analyze a specific size population. The instrument takes an average of all the objects in the image and determines the average cell size. Then it sets limits above and below that average size based upon the adjustable settings.

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

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 adjustments to certain parameters, and those may be optimized by the user.

We have only tested the Countess™ instrument with trypan blue.

So far, we have not found any alternative dyes that can be used with the Countess™ instrument. We have tested eosin Y, eosin B, and nigrisin. However, we have determined that a lower concentration of trypan blue (0.1%) does work with the instrument, which can help to minimize contact with trypan blue. 

The Countess™ instrument comes precalibrated. You have to recalibrate it if you update the firmware on the instrument. You also have to recalibrate the instrument if you decide to use a different brand or concentration of trypan blue than is supplied with the kit. Calibration simply sets the background to the correct blue color and takes approximately 2–3 minutes. Other than these two situations, you do not have to calibrate the instrument.

We have tried trypan blue concentrations from 0.05–0.6%, and they all still work, as long as the instrument is recalibrated for that concentration of trypan blue.