Cell counting using a hemocytometer is a cornerstone of in vitro and in vivo experiments. It allows for precision and reproducibility in your studies and enhances the reliability of your results. Whether your research focuses on microbiology, hematology, oncology, or other diseases, if it involves cell culture, accurate manual counting using a hemocytometer is essential.
This protocol covers how to use a hemocytometer for cell counting, from preparing your sample to calculating cell viability using trypan blue staining. It describes how to count cells with a Neubauer chamber (but the same protocol applies to a Burker chamber) and the cell counting formulas to use for percentage viability based on live-dead staining using trypan blue (relative cell viability) so you can confidently use a hemocytometer in your experiments.
If using a glass hemocytometer and coverslip, clean with alcohol before use.
The presence of Newton's refraction rings under the coverslip indicates proper adhesion.
If using a disposable hemocytometer (for example, INCYTO DHC-N01), simply remove from the packet before use.
Aseptic technique prevents contamination of cell cultures and reagents by microorganisms. Our aseptic technique video protocol shows you how to sterilize work areas and use appropriate sterile handling techniques, personal protective equipment, and good hygiene.
Table 1. The volume of DPBS and trypsin-EDTA required for trypsinization of adherent cells.
T-flash (cm2) | DPBS (mL) | Trypsin-EDTA (mL) | FBS containing media required to neutralize trypsin |
25 | 2 | 2 | 6 |
80 | 3 | 3 | 9 |
175 | 5 | 5 | 15 |
Gently swirl the flask to ensure the cells are evenly distributed.
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.
Take 100 µL of cells into a new Eppendorf tube.
Using a pipette, take 100 µL of Trypan Blue-treated cell suspension and apply to the hemocytometer.
Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.
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).
Figure 1. Hemocytometer diagram indicating one of the sets of 16 squares that should be used for counting.
Move the hemocytometer to the next set of 16 corner squares and carry on counting until all 4 sets of 16 corners are counted.
Take the average cell count from each of the sets of 16 corner squares.
Multiply
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, for example: