Trypan Blue is the stain most commonly used to distinguish viable from nonviable cells. Viable cells exclude the dye, while nonviable cells absorb the dye and appear blue. Cells should be in suspension as single cells in buffered saline before counting. Trypan Blue has a higher affinity for serum protein than for cellular proteins, so suspending cells in medium containing serum will generate a dark background.
Cell Core apparatus (Larminar flow hood with asiration tube and Bensen burner, Pastear pipet, Microscope, ...)
Trypan Blue stock solution.
Procedure
Trypsinize the cells and break the clumps to single cells.
Aseptically withdraw a sample of the cell suspension and prepare a series of dilutions, as required, in PBS. The optimal concentration of cells for counting is 5-10x10 5 cells/ml (50-100 cells per large square) after dilution in the Trypan Blue solution.
Add Trypan Blue stock so that the final concentration is 0.1%. Or dilute the Trypan Blue stock to 0.2% solution and add to cell suspension at 1:1.
After being stained with Trypan Blue, the cells should be counted within 3 minutes: after that time the cells will begin to take up the dye.
Using a Pasteur pipette, withdraw a small amount of the stained cell suspension and place the tip of the pipette onto the slot of a clean hemacytometer with coverslip. The cell suspension will pass under the coverslip by capillary action. Fill the opposite chamber with the second diluted sample. Do not overfill. Do not lift or move the coverslip.
Place the hemacytometer on the stage of an inverted microscope. Adjust focus and power until a single counting square fills the field.