Trypan blue will stain dead or dying cells. Viable cells are able to repell the dye and do not stain. Note: Trypan blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting.
Supplies & Equipment:
Eppendorf tubes (1.5 ml)
Micropipet (10µl)
Hemocytometer
Reagents:
HBSS (Hanks' Balanced Salt Solution)
sterile Trypan blue solution 0.4% (Sigma T-8154)
Procedure:
Prepare a cell suspension in HBSS
Transfer into Eppendorf tube:
0.5 ml of 0.4% Trypan blue solution
0.3 ml of HBSS
0.2 ml of cell suspension in HBSS (= dilution 1 : 5)
Allow to stand for 5 to 15 minutes
Note: after prolonged incubation, viable cells start to take up dye as well.
Pipet 10µl of this mix into cover-slipped chambers of hemocytometer
Note: avoid cell clusters by pipetting up and down.
Count viable and non-viable cells
Note: for optimal results, adjust cell density to 20-50 cells / square.
Calculations:
cells/ml: the number of cells per quadrant equals 104 cells / ml
(e.g. 50 cells per quadrant = 0.50 million cells / ml)
total cells: cells / ml x original volume
(e.g. 5 million cells in 10 ml)
cell viability (%): total viable cells (unstained) / total cells (stained and unstained) x 100 (e.g. 25 stained cells per quadrant: 50% viability)
