How to Measure Cell Viability

In most biological experiments involving cell culture, a critical step includes knowing which cells are alive and which are dead in your sample. Dye exclusion is the most popular method of measuring cell viability. Dye exclusion is based on the theory that live cells contain intact membranes and dead cells do not, thus dead cells absorb the dye into the cytoplasm. There are different dyes and protocols and the methods you choose should be based on cost, sensitivity, ease and length of procedure. Trypan blue is a common dye used to measure cell viability because the procedure can be done in five to 10 minutes and costs only the amount of the dye reagent.

Things You'll Need

• 0.4% trypan blue reagent
• Hemacytometer
• Cover slip
• Micropipette
• Pipette tips
• Phosphate buffered saline (PBS)
• Light microscope

Show More

Instructions

• 1

  Obtain a pellet of the cells you're measuring.

• 2

  Resuspend the cell pellet in PBS to get approximately 5 x 105 cells/ml. PBS is used because viability measurements are more accurate when cells are in a serum-free environment; serum proteins may also stain and give you misleading results.

• 3

  Add equal parts of cell suspension in PBS to equal parts of 0.4% trypan blue dye to obtain a 1 to 2 dilution (example: 100 ul of cells to 100 ul of trypan blue) and mix by pipetting up and down.

• 4

  Incubate mixture for less than three minutes at room temperature. If cells are counted after approximately five minutes, viability will be inaccurate due to cell death.

• 5

  With the cover slip already in place, fill each side of a hemacytometer counter with the cell suspension. Typically, each side will take 10 to 20 ul.

• 6

  Place the hemacytometer on the stage of a light microscope and focus onto the cells.

• 7

  Each side of the hemacytometer contains multiple squares. Count the total number of cells in one square of the hemacytometer. Then count the number of non-viable (blue) and viable (clear) cells separately, in the same square.

• 8

  Calculate the percentage of viable cells in the square by dividing the number of viable cells by the number of total cells and multiplying by 100. Do this for multiple squares on the hemacytometer to obtain an average viability measurement.