Diphenyl Blue

Diphenyl Blue (Trypan Blue) is a cell active dye, the most commonly used dye for the identification of dead cells, of en used to test cell membrane integrity and cell viability. Diphenyl Blue staining is one of the methods for tissue and cell culture. When cells are deactivated or have incomplete cell membranes, Diphenyl Blue can stain them Blue. Normal living cells with intact cell membranes reject Diphenyl blue and do not stain them blue. However, macrophages are capable of phagocytosis of Diphenyl Blue, so it can be used as a living stain for macrophages^[1]. In Vitro: Preparation of Trypan Blue working solution
1.1 Preparation of the stock solution
Dissolve 10 mg of Trypan Blue in 100 mL 0.85% Nacl to obtain 0.4% Trypan Blue.
Note: It is recommended to store the stock solution at -20℃ -80℃ away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of Trypan Blue working solution
Dilute the stock solution in 0.85% Nacl to obtain 0.04% of Trypan Blue working solution.
Note: Please adjust the concentration of Trypan Blue working solution according to the actual situation.
Cell staining
2.1 For suspension cells, centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells, discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.2 Add 1 mL of rypan Blue working solution, and then incubate at room temperature for 5 minutes.
2.3 Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
2.4 Wash twice with PBS, 5 minutes each time.
2.5 Resuspend cells with serum-free cell culture medium or PBS, and then detect by fluorescence microscope.