Trypan Blue Dye Exclusion Assay

Cell Viability Testing with Trypan Blue Exclusion

The trypan blue dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that viable cells with intact cell membranes are impermeable to polar dyes, such as trypan blue, whereas in dead cells with porous membranes, trypan blue can readily penetrate and stain the cytoplasm blue. Upon analysis by light microscope, the number of stained cells can be examined against the total cell population. The number of stained cells will represent the percentage of dead cells in the entire population.

The following procedure will enable you to accurately determine cell viability in a cell suspension.

Materials:

Cells in suspension
Trypan Blue, sodium salt *UltraPure grade* (AAT Bioquest Cat No. 2452)
PBS buffer (pH 7.2 – 7.3) [for recipe, click here]
Centrifuge
Hemocytometer
Coverslips
Two key differential counter

Method

Centrifuge an aliquot of cell suspension for 5 minutes at 100 x g, and then discard the supernatant.
1. Note: The size of the aliquot depends on the approximate number of cells present, and should contain an appropriate number of cells to count in a hemocytometer when suspended in 1 mL PBS and then diluted again by mixing with 0.4% trypan blue.
2. Note: For the adherent cells, in order to detach from the plate, trypsinize the cells and resuspend it in medium to stop the process. Centrifuge the mixture, remove the supernatant and then follow next steps.
Re-suspend cells in 1 mL PBS buffer (pH 7.2 – 7.3).
Prepare a 0.4% trypan blue solution in PBS buffer, pH 7.2 to 7.3.
- For example: To make a 1 mL 0.4% trypan blue solution, dissolve 4 mg of trypan blue in 1 mL PBS buffer (pH 7.2 – 7.3)
Take 100 µL of cells into a centrifuge tube and add 400 µL of 0.4% trypan blue solution, and incubate mixture at room temperature for 5 minutes.
Add 100 µL of trypan blue treated cells and apply to the hemocytometer and place a coverslip on the hemocytometer chambers and carefully fill chambers with trypan blue treated cells.
- Note: Be careful not to under- or over-fill the chambers. Avoid having any bubbles in the counting region.
Incubate the cells with the hemocytometer at room temperature for 1 to 2 minutes.
- Note: For longer incubations it is recommended to place the hemocytometer in a humid chamber. Do not exceed incubation times longer than 30 minutes.
Place the hemocytometer under microscope and count the cells in four '1 x 1 mm' squares (# squares=16) of one chamber and determine the average number of cells per square.
- Note: Accurate cell count numbers in a '1 x 1 mm' square should range from 20 to 50 cells. If a cell count is greater than 50 is observed, then dilute the cell suspension further. If less than 20 cells are counted, then use undiluted sample.
Count the number of blue staining cells and the number of total cells. Cell viability should be at least 95% for healthy log-phase cultures.

Formulas

To calculate cell viability use the following formula:

To calculate the number of viable cells per mL of culture take the average values of the cell counts for each of the 16 corner squares, and use the following formula:

Note: Only count the cells which are not stained with Trypan Blue (viable cells).

Note: Remember to correct for the dilution factor.

Product Ordering Information

Document: 02.0120.190613r1

Last updated Fri Aug 29 2025