- If you are using a glass hemocytometer and a coverslip, clean it with alcohol before use. Moisten the coverslip with water and place it on the hemocytometer. The presence of Newton’s rings of refraction under the coverslip indicates adequate adhesion.
- If you use a disposable hemocytometer (for example, INCYTO DHC-N01), simply remove it from the package before use.
Preparing cell suspension
The aseptic technique avoids contamination of cell cultures and reagents by microorganisms. Watch our Aseptic Technique video protocol showing you how to sterilize work areas and use proper sterile handling techniques, personal protective equipment, and good hygiene.
- Gently swirl the flask to ensure that the cells are evenly distributed.
- Before cells have a chance to settle, remove 0.5 ml of cell suspension with a sterile 5 ml pipet and place it in an Eppendorf tube.
- Place 100 µL of cells into a new Eppendorf tube and add 400 µL of 0.4% trypan blue (0.32% final concentration). Mix gently.
- Using a pipette, take 100 µL of trypan blue-treated cell suspension and apply it to the hemocytometer. If using a glass hemocytometer, very gently fill both chambers under the coverslip, allowing the cell suspension to be removed by capillary action. If using a disposable hemocytometer, pipet the cell suspension into the well of the counting chamber, allowing capillary action to draw it in.
- Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.
- Using a manual counter, count unstained live cells (live cells do not absorb trypan blue) in a set of 16 squares. When counting, use a system where cells are only counted when placed within a square or on the lower or right boundary line. Following the same guidelines, trypan blue stained dead cells can also be counted for a viability estimate if necessary.
- Move the hemocytometer to the next set of 16 corners and continue counting until all 4 sets of 16 corners are counted.
To calculate the number of viable cells / mL:
- Take the average cell count of each of the sets of 16 corner squares.
- Multiply by 10,000 (104).
- Multiply by 5 to correct the 1: 5 dilution of the trypan blue addition.