Trypan Blue

Trypan Blue:

Exclusion The following procedure will enable you to accurately determine the cell viability. Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the hemacytometer. If cells take up trypan blue, they are considered non-viable.

1.        Determine the cell density of your cell line suspension using a hemacytometer.

2.        Prepare a 0.4% solution of trypan blue in buffered isotonic salt solution, pH 7.2 to 7.3 (i.e., phosphate-buffered saline).

3.        Add 0.1 mL of trypan blue stock solution to 1 mL of cells.

4.        Load a hemacytometer and examine immediately under a microscope at low magnification.

5.        Count the number of blue staining cells and the number of total cells. Cell viability should be at least 95% for healthy log-phase cultures. Remember to correct for the dilution factor.

To calculate the number of viable cells per mL of culture, use the formula below.

Live cell count/ Total cell count =Viability

Determine total viable cell yield using the formula below.

Viable cell count/ Quadrants counted x Dilution factor x Hemocytometer factor x Current volume (mL) = Viable cell yield.