Cell viability measurement and counting Catalog Number HPR116

Cell viability measurement and cell counting are determined by trypan blue (0.05% in D-PBS 1X) exclusion test.

1. Transfer 900 µL of trypan blue solution (0.05% in D-PBS 1X) in a 5 mL polystyrene round bottom tube.
2. Prepare a cell counting chamber (e.g., Nageotte chamber). by carefully cleaning with lens paper. The coverslip should also be cleaned and placed over the counting surface prior to putting on the cell suspension.
3. Gently homogenize the cell suspension by manual swirling.
4. Dilute 100 µL of the cell suspension in the 900 µL of trypan blue solution.
5. Gently homogenize the obtained cell suspension.
6. Pipette around 100 µL of cell suspension between the mirror-like polished surface and coverslip. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is fully covered.
7. Proceed to observation under microscope. Count living and dead cells on at least four rows distributed throughout the cell counting chamber. Living cells exclude the dye, dead cells take up the dye and appear blue. If the total number of cells is quite different from one row to another, count one or two more rows.
   Note: Thawed differentiated HepaRG® cells can form clusters. It’s necessary to count all the cells including those forming the clusters.
8. Determine the average number of viable cells and dead cells per row.
9. Determine percentage of cell viability.
10. Calculate the cell concentration in million cells per ml.

Sample calculation with a Nageotte chamber

Percentage of viable cells: (Number of viable cells)/(Number of viable cells + number of dead cells) X 100

Million cells per ml: Number of viable cells per row X 10 (dilution factor in trypan blue) X 800 (parameter relating to the Nageotte cell)

Total number of cells: Million cell/mL Cell concentration in million cells / ml X Total volume of cell suspension