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:Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Preparation of Trypan Blue Working Solution
1.1 Prepare 0.4% trypan blue solution with pure water. For example, dissolve 400 mg trypan blue in 100 mL of pure water.
1.2 Dilute the stock solution with preheated serum-free cell culture medium or PBS to prepare 0.04% trypan blue working solution.
Note: Please adjust the concentration of trypan blue working solution according to the actual situation, and prepare it fresh each time you use it.

2. Cell Staining
2.1 Suspension Cells: Collect cells by centrifugation, wash twice with PBS, 5 minutes each time.
Adherent Cells: Discard the culture medium, wash twice with PBS, 5 minutes each time.
2.2 Add trypan blue working solution and incubate at room temperature for 5 minutes.
2.3 Discard the staining solution, wash cells twice with PBS, 5 minutes each time.
2.4 After resuspending the cells in serum-free culture medium or PBS, cell viability can be accurately quantified by direct counting under a microscope or by counting after taking photographs under a microscope.