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        Trypan blue viability test

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        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)

         

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