Hoechst 33258 is a marker dye in Hoechst series. Hoechst is A live nuclear marker dye. Hoechst binds to the grooves in the DNA double strand, which tends to be A/ T-rich DNA strand. Although it binds to all nucleic acids, the A/ T-rich double strand DNA significantly enhances fluorescence intensity Therefore,Hoechst dye can be used for living cell labeling. The fluorescence intensity of Hoechst dye increases with the increase of pH of solution^[1].

IC50: 51.31±4.56 μM (HeLa cell), 32.43±3.27 μM (HL60 cell), 15.42 ± 2.16 μM (U937 cell)^[1]

General Protocol
Preparation of Hoechst working solution
1.1 Preparation of the stock solution
Dissolve 10 mg of in 5 mL DMSO
Note: It is recommended to store the stock solution at 4℃ or -20℃ away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of Hoechst working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain final concentration 10 μg/mL Hoechst working solution.
Note: Please adjust the concentration of Hoechst working solution according to the actual situation.
1.Cell staining
2.1 Suspension cells（6-well plate）
a. Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time. The cell density is 1×10⁶/mL.
b. Add 1 mL of working solution, and then incubate at room temperature for 3-10 minutes.
c. Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
d. Wash twice with PBS, 5 minutes each time.
e. Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslip from the medium and aspirate excess medium.
c. Add 100 μL of working solution, gently shake it to completely cover the cells,and then incubate at room temperature for 3-10 minutes.
d. Wash twice with medium, 5 minutes each time.Observation by fluorescence microscopy or flow cytometry.

Precautions
1. Please adjust the concentration of Hoechst working solution according to the actual situation.
2. This product is for R&D use only, not for drug, household, or other uses.
3. For your safety and health, please wear a lab coat and disposable gloves to operate.

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

* 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 (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Wang XJ, et al. Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through inductionof apoptosis and autophagy. Bioorg Med Chem Lett. 2012 Oct 1;22(19):6297-300.  [Content Brief]

  [2]. Stolić I, et al. Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene. Eur J Med Chem. 2011 Feb;46(2):743-55.  [Content Brief]