Diphenyl Blue (Direct Blue 14)

This protocol provides a guide only and should be modified according to your specific needs.

Ⅰ. Preparation of Diphenyl Blue (Trypan Blue) Dye Solution

1. Prepare diphenyl blue (trypan blue) stock solution: Dissolve 400 mg of trypan blue in 100 mL of 0.85% Nacl to obtain a 0.4% trypan blue stock solution.

Note: It is recommended to store the stock solution in aliquots at -20°C-80°C in the dark, and avoid repeated freeze-thaw cycles.

2. Prepare diphenyl blue (trypan blue) working solution: Use serum-free cell culture medium or PBS to dilute the stock solution to prepare trypan blue working solution with a concentration of 0.04%.

Note: Please adjust the concentration of the trypan blue working solution according to the experimental needs, and prepare it now.

Ⅱ. Cell staining

1. Suspended cells: Collect cells by centrifugation, add PBS to wash twice, 5 minutes each time.

Adherent cells: remove medium and add trypsin to digest cells. Discard the supernatant after centrifugation, add PBS to wash twice, 5 minutes each time.

2. Add 1mL trypan blue working solution and incubate for 5 minutes at room temperature.

3. Centrifuge at 400g for 3-4 minutes at 4°C, discard the supernatant, add PBS to wash the cells twice, 5 minutes each time.

4. Resuspend the cells with 1 mL of serum-free medium or PBS. Cell viability was calculated by counting under a microscope or after taking pictures under a microscope^[3].

Cell survival rate (%)=total number of live cells/(total number of live cells+total number of dead cells)*100%

3. Matters needing attention:

1. The staining time should not be too long, otherwise the living cells will gradually accumulate the dye and become stained, resulting in low test results.

2. If there is precipitation in the dye solution before dyeing, it needs to be filtered to remove the precipitation before use.

3. There is a potential carcinogenic risk. For your safety and health, please wear a lab coat and disposable gloves for operation.

References:

[1]. Warren Strober.Trypan Blue Exclusion Test of Cell Viability. 2015 Nov 2;111:A3.B.1-A3.B.3. doi: 10.1002/0471142735.ima03bs111.