Hoechst 33258 trihydrochloride (Bisbenzimide) is a benzimidazole anti-filarial agent. it is fluorescent when it binds to certain nucleotides in DNA, thus providing a tool for the study of DNA replication. it also interferes with mitosis.

HeLa cells:51.31±4.56 μM (IC50), HL-60 cells:32.43±3.27 μM (IC50), U937 cells:15.42±2.16 μM (IC50)

Solution preparation
a. Preparation of stock solution: Dissolve 10 mg in 5 mL DMSO; (It is recommended to store at -20 ℃ or -80 ℃ in the dark after aliquoting)
b. Preparation of Hoechst working solution
Dilute the stock solution and dissolve it in serum-free cell culture medium or PBS to obtain a Hoechst working solution with a final concentration of 10 μg/mL; (Select the appropriate working solution concentration according to experimental requirements and prepare it for immediate use)
c. Cell staining
1. Suspended cells (6-well plate)
a. Centrifuge at 1000 g for 3-5 minutes at 4℃, and then discard the supernatant. Wash twice with PBS for 5 minutes each. The cell density is 1×106/mL;
b. Add 1 mL of working solution and incubate at room temperature for 3-10 minutes;
c. Centrifuge at 400 g for 3-4 minutes at 4℃, and discard the supernatant;
d. Wash twice with PBS, 5 minutes each time;
e. Resuspend cells with serum-free cell culture medium or PBS. Observe with fluorescence microscope or flow cytometer.
2. Adherent cells
a. Culture adherent cells on sterile coverslips;
b. Remove coverslips from culture medium and aspirate excess culture medium;
c. Add 100 μL working solution, shake gently to completely cover cells, and incubate at room temperature for 3-10 minutes;
d. Wash twice with culture medium, 5 minutes each time. Observe with fluorescence microscope or flow cytometer.