Trypan blue viability test

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)