Trypan Blue Exclusion
- Determine the cell density of your cell line suspension using a hemacytometer.
- Prepare a 0.4% solution of trypan blue in buffered isotonic salt solution, pH 7.2 to 7.3 (i.e., phosphate-buffered saline).
- Add 0.1 mL of trypan blue stock solution to 1 mL of cells.
- Load a hemacytometer and examine immediately under a microscope at low magnification.
- 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.
% viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100
To calculate the number of viable cells per mL of culture, use the formula below. Remember to correct for the dilution factor.
Number of viable cells × 104 × 1.1 = cells/mL culture