Trypan blue viability test

Outline:

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:

1. Prepare a cell suspension in HBSS
2. 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)
3. Allow to stand for 5 to 15 minutes
   Note: after prolonged incubation, viable cells start to take up dye as well.
4. Pipet 10µl of this mix into cover-slipped chambers of hemocytometer
   Note: avoid cell clusters by pipetting up and down.
5. Count viable and non-viable cells
   Note: for optimal results, adjust cell density to 20-50 cells / square.
6. 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)