Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell Trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue colour under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method.
Background & chemistry
Uses of trypan blue
Protocol for vital staining
Place a suitable volume of a cell suspension (20-200 μL) in appropriate tube add an equal volume of 0.4% Trypan blue and gently mix, let stand for 5 minutes at room temperature. Place 10 μl of stained cells in a hemocytometer and count the number of viable (unstained) and dead (stained) cells. Calculate the average number of unstained cells in each quadrant, and multiply by 2*104 to find cells/ml. The percentage of viable cells is the number of viable cells divided by the number of dead and viable cells.
- Chapter "Detection of Caspase Activation Combined with Other Probes of Apoptosis", Eurekah Bioscience Collection, NCBI bookshelf
- Protocol for use of the dye from Northwestern University (PDF)
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