Diphenyl Blue  (Synonyms: Direct Blue 14; Trypan 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].

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.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

960.81

C₃₄H₂₄N₆Na₄O₁₄S₄

72-57-1

Solid

Light brown to black

CC1=CC(C2=CC=C(/N=N/C3=C(S(=O)(O[Na])=O)C=C4C=C(S(=O)(O[Na])=O)C=C(N)C4=C3O)C(C)=C2)=CC=C1/N=N/C5=C(S(=O)(O[Na])=O)C=C6C=C(S(=O)(O[Na])=O)C=C(N)C6=C5O

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture and light

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

• [1]. Daly, M. L., DeRosa, C. A., Kerr, C., Morris, W. A., & Fraser, C. L. (2016). Blue thermally activated delayed fluorescence from a biphenyl difluoroboron β-diketonate. RSC Advances, 6(85), 81631–81635. doi:10.1039/c6ra18374c