Cell Counting Procedure
Step 5. Vortex the target cell suspension and mix 1:1 with 0.2% trypan blue
Step 6. Pipette 10 microliters of cell sample into the hemacytometer
Step 7. Wait 30 seconds for the cells to settle
Manually Count Cells in Sample
Step 8. Place the hemacytometer under a microscope using a 10x or a 20x objective
Step 9. Focus both onto the grid pattern and the cell particles, and count the total number of cells found in 4 large corner squares. If cells are touching the 4 perimeter sides of a corner square, only count cells on 2 sides, either the 2 outer sides or 2 inner sides
Step 10. Count the live cells (without trypan blue) and dead cells (with trypan blue)
Cell Calculations & Cleaning of Hemacytometer
Step 11. Obtain cell concentration (cells/ml) by multiplying the dilution factor by the total number of cells, divide by the # of corner squares counted, and multiply by 104.
Step 12. Clean hemacytometer and glass cover slip with 70% EtOH
Similar counting procedures can also be performed using the Nexcelom disposable hemacytometer.