Various cellular manipulations, including passaging, freezing, and isolation of primary tissues, can lead to cell death. To determine the number of surviving cells in a population of cells, the trypan blue dye rejection assay can be used (Phillips 1973). This experiment was derived from the Guide to Cellular Experiments (previous volume) by Peitang Huang.

Operation method

Vibrant Coloring

Principle

Various cellular manipulations, including passaging, freezing, and isolation of primary tissues, can lead to cell death. In order to determine the number of viable cells in a population, the trypan blue dye rejection assay is used (Phillips 1973). Normal healthy cells repel the dye, but the loss of cell membrane integrity allows TPB to diffuse into the cell. The dye rejection assay is a crude method of estimating cell viability and is unable to distinguish a 10%-20% difference in viability. In addition, cells that reject the dye may not adhere to the wall or may not survive or multiply for a longer period of time.

Move

1) Trypsin-treated cells (see "Trypsin Treatment of Isolated Cells" above), aseptically take 0.5 ml of cells and dilute them with PBS to 2X10 5 ^~4X105 per ml.

2) Aseptically add 0.5 ml of the above cell dilution solution to another tube, and add 0.5 ml of Taipan blue solution (0.4%).

3) The cells should be kept in the dye for not less than 3 minutes and not more than 10 minutes, and the cell suspension containing the dye should be taken by capillary pipette and sucked into the counter by capillary action.

4) Count a total of at least 500 cells, with blue cells counted separately. Record the frequency of blue cells that do not reject the dye.

5) Count the percentage viability of the cells according to the number of cells that do not reject the dye.

For example: trypsinize monolayer cells and resuspend them in 5 ml of culture medium, take 0.5 ml of cells and mix with 4.5 ml of PBS, take 0.5 ml of cells suspended in PBS and add it to another small tube, then add 0.5 ml of Taipan blue solution and mix well. The above suspension was added to a counter and a total of 540 cells were counted, 62 cells did not reject the dye and showed blue color, with an activity percentage of 88.5%.

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