How to Count Cells Using a Hemocytometer

Option A: Dilution and Staining of Freshly Prepared PBMCs

1. Prepare red cell lysis stain (50 ul 6% acetic acid + 50 uL Trypan Blue).
2. Make 1:10 dilution (5 uL of PBMC into 45 uL HBSS or PBS).
3. Transfer 20 ul each of dilution to two separate new microfuge tubes.
4. Add 20 ul red cell lysis stain (step 1) to one microfuge tube containing 20 ul of dilution from step 2.
5. Add 20 ul Trypan Blue to one microfuge tube containing 20 ul of dilution from step 2.
6. Incubate for at least 1 min.

NOTE: do not count cells that have been in staining buffers for longer than 15 min as non-specific staining can occur.

Option B: Dilution and Staining of Thawed Cells or Purified Subsets

1. Make 1:10 dilution (5 uL of cell solution into 45 uL HBSS or PBS).
2. Transfer 20 ul of the dilution to a separate microfuge tube.
3. Add 20 ul Trypan Blue to the microfuge tube.
4. Incubate for at least 1 min.

NOTE: do not count cells that have been in staining buffers for longer than 15 min as non-specific staining can occur.

1. Pre-clean hemocytometer and cover slip with 70% EtOH.
2. Moisten the coverslip with water and allow to adhere to the hemocytometer. Rings will be visible when proper adhesion is attained.
3. Gently mix stained PBMC or cell solutions (prepared above) by flicking tube.
4. If counting freshly prepared PMBC: withdraw 10 uL of the PMBC cell solution containing red cell lysis buffer and slowly dispense at the sample introduction point. The counting chamber should fill by capillary action. Rotate the hemocytometer, withdraw 10 uL of the PMBC cell solution stained with Trypan Blue and slowly dispense at the sample introduction point on the opposite side of step 4.
5. If counting thawed cells or cell subsets: load both sides of the hemocytometer as described in step 4 with 10 uL of the same stained cell solution.
6. Allow samples to settle for 30 seconds so all cells are on the same plane.

1. Gently clean microscope lens with a Kimwipe and 70% EtOH. This only needs to be done once per day of use.
2. Place hemocytometer onto microscope stage.
3. Adjust microscope objective to 10X and adjust focus so that the cells are clearly visible.
4. There should be approximately 50-100 cells within each of the four outside squares (Figure 1a, blue boxes). If the estimated number of cells is outside this range, prepare a new sample dilution.
5. Starting with the upper left outside square, use the hand-tally counter to record the count of the unstained (viable) cells. As shown in Figure 1b, do not count cells that lie on the outer two perimeters, but do count those on the inner two perimeters.
6. Repeat the cell count for the same square, counting the total number of both stained cells (non-viable) and unstained cells (viable) in each square.
7. Repeat step 5 and/or 6 for each outside square in a clockwise direction, recording the total and resetting the hand-tally counter after each count.

Figure 1. Hemocytometer gridlines used for cell counting

8. Determine the average # of viable cells by adding each individual square box count and dividing by 4.
9. Determine the average # of viable and non-viable cells by adding each individual square box count and dividing by 4.
10. Cell concentration can be determined using the following equation:

Total # cells/mL = average viable cell # (step 8) x dilution factor x 10⁴

Example: Average viable cell # square (step 8) = 35 with 1:10 dilution:

Total # cells/mL = 35 x 10 x 10⁴ = 3.5 x 10⁶ cells/mL

11. Cell viability can be calculated by the following equation:

Average viable cell # (step 8)/average viable and non-viable cell# (step 9)

Example: Average viable cell # (step 8) = 35, average total (viable and non-viable) cell # = 38:

Cell viability = 35/38 (x100) = 92.1%

12. Record cell count and viability on data entry form.
13. Clean the hemocytometer and coverslip carefully with 70% ethanol.