Gently swirl the flask to ensure the cells are evenly distributed.
Before the cells have a chance to settle, take out 0.5 mL of cell suspension using a 5 mL sterile pipette and place in an Eppendorf tube.
Take 100 µL of cells into a new Eppendorf tube and add 400 µL 0.4% Trypan Blue (final concentration 0.08%). Mix gently.