Counting cells using a hemocytometer

Protocol to obtain a viable cell count from suspension cells using a hemocytometer (6:51 minutes).

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Preparing hemocytometer

1. If using a glass hemocytometer and coverslip, clean with alcohol before use. Moisten the coverslip with water and affix to the hemocytometer. The presence of Newton's refraction rings under the coverslip indicates proper adhesion.
2. If using a disposable hemocytometer (for example, INCYTO DHC-N01), simply remove from the packet before use.
   
   

Preparing cell suspension

Aseptic technique prevents contamination of cell cultures and reagents by microorganisms. Watch our aseptic technique video protocol that shows you how to sterilize work areas and use appropriate sterile handling techniques, personal protective equipment, and good hygiene.

1. Gently swirl the flask to ensure the cells are evenly distributed.
2. Before the cells have a chance to settle, take out 0.5 mL of cell suspension using a 5 mL sterile pipette and place in an Eppendorf tube.
3. Take 100 µL of cells into a new Eppendorf tube and add 400 µL 0.4% Trypan Blue (final concentration 0.32%). Mix gently.

Table 1. The volume of DPBS and trypsin-EDTA required for trypsinization of adherent cells.

Counting

1. Using a pipette, take 100 µL of Trypan Blue-treated cell suspension and apply to the hemocytometer. If using a glass hemocytometer, very gently fill both chambers underneath the coverslip, allowing the cell suspension to be drawn out by capillary action. If using a disposable hemocytometer, pipette the cell suspension into the well of the counting chamber, allowing capillary action to draw it inside.
2. Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.
3. Using a hand tally counter, count the live, unstained cells (live cells do not take up Trypan Blue) in one set of 16 squares (Figure 1). When counting, employ a system whereby cells are only counted when they are set within a square or on the right-hand or bottom boundary line. Following the same guidelines, dead cells stained with Trypan Blue can also be counted for a viability estimate if required.
4. Move the hemocytometer to the next set of 16 corner squares and carry on counting until all 4 sets of 16 corners are counted.
   
   

Viability

To calculate the number of viable cells/mL:

1. Take the average cell count from each of the sets of 16 corner squares.
2. Multiply by 10,000 (10⁴).
3. Multiply by 5 to correct for the 1:5 dilution from the Trypan Blue addition.

The final value is the number of viable cells/mL in the original cell suspension.

Example: 

• If the cell counts for each of the 16 squares were 50, 40, 45, 52, the average cell count would be:
• (50 + 40 + 45 +52) ÷ 4 = 46.75
• 46.75 x 10,000 (10⁴) = 467,500
• 467,500 x 5 = 2,337,500 live cells/mL in original cell suspension

To calculate viability:

If both live and dead cell counts have been recorded for each set of 16 corner squares, an estimate viability can be calculated.

1. Add together the live and dead cell count to obtain a total cell count.
2. Divide the live cell count by the total cell count to calculate the percentage viability.

Example:

• Live cell count: 2,337,500 cells/mL
• ​Dead cell count: 50,000 cells/mL
• 2,337,500 + 50,000 = 2,387,500 cells
• 2,337,500 ÷2,387,500 = 97.9% viability

Figure 1. Hemocytometer gridlines.
​​Hemocytometer diagram indicating one of the sets of 16 squares that should be used for counting.

Webinar transcript

Counting cells allows the accurate determination of cell numbers, and therefore, consistency between experiments. This video will outline the procedure for counting both suspension and adherence cells using a hemocytometer.

Before commencing work, thoroughly spray the inside of the laminar flow safety cabinet with disinfectant and wipe clean with tissue. Dispose of used tissue in the appropriate waste bin. Next, spray the inside of the hood with 70% ethanol and wipe clean with tissue. The hood is now clean and ready to use. Remove cell culture media and trypsin from the fridge, and place in a humidified, 37-degree C, carbon dioxide incubator to warm. Meanwhile, look at the cells to be counted using a microscope to check for any visual signs of bacterial and fungal contamination. Spray media bottles and pipettes with 70% ethanol before placing in the laminar flow safety cabinet.

For suspension cells, gently agitate the flask to ensure the cells are well mixed. Before they get a chance to settle, take an 0.5-milliliter sample of cell suspension and pipet into a sterile Eppendorf tube. For adherence cells, remove existing media, wash with room temperature PBS, and add trypsin EDTA to detach the cells. Refer to Table One for the volumes of PBS and trypsin required. Incubate the cells for two to five minutes in the humidified 37 degrees C, carbon dioxide incubator. The incubation time will need to be optimized for the cell type. When all cells are detached, neutralize the trypsin EDTA with warm serum-containing growth media appropriate to the cells and culture. Refer to Table One for the required volumes.

Resuspend the cells by gently pipetting the cell suspension up and down three times and transfer them into a 50-milliliter tube. Centrifuge the cell suspension for five minutes at 1,000 revolutions per minute at room temperature. Remove the supernatant using a sterile serological pipette and re-suspend the cell pellet with 37-degree C serum containing growth media to the original volume of the starting culture. Using a five-milliliter sterile pipette, take a 0.5-milliliter sample of cell suspension, and transfer into a sterile Eppendorf tube.

To begin counting, prepare the disposable hemocytometer. Using a P2000 Gilson Pipette, take 100 microliters of suspension, and add to 400 microliters of trypan blue, note, 0.08%, and mix well. Take 100 microliters of the trypan blue cell suspension mix, and carefully pipette a drop of the suspension into the well of the counting chamber, allowing capillary action to draw the sample in. Take care not to overfill the counting chamber. View the counting area under a 10 times magnification using an inverted microscope. Using the microscope, focus on one of the four by four grids on the hemocytometer and count the cells that are negative for trypan blue. These ones are viable. Also, make note of how many cells were positive for trypan blue. These cells are dead, and this number can be used later to calculate the percentage viability of the culture if required.

When counting, employ a system whereby cells are only counted when they are within a square, or on the right-hand or bottom boundary line. Record the number of cells counted in this set of 16 squares and move the hemocytometer until all four sets of 16 squares on the hemocytometer have been counted, and their values recorded. To calculate the cell concentration, take the average number of viable cells in the four sets of 16 squares and multiply by 10,000 to get the number of cells per milliliter. Then, multiply this by five to correct for the one in five dilution from the trypan blue addition. This final value is the number of viable cells per milliliter in the original cell suspension.

For example, if your viable cell count is 200,000 cells per milliliter in a volume of 20 milliliters and you want to see 10,000 cells into the new flask, then you need to transfer one milliliter of your cell suspension into the new flask. Transfer the required volume of cell suspension into the new flask. Top up with media and put into the incubator.