Hoechst 33342 (bisBenzimide H 33342), is a Benzimidazole fluorescent dye and a Cell permeable fluorescent DNA stain; binds minor groove of AT-rich regions. HOE 33342 trihydrochlorde is used to quantify DNA in viable cells.

1. Preparation of Hoechst 33342 working solution
1. Preparation of stock solution
Please use DMSO to prepare 1 mg/mL stock solution of Hoechst 33342.
Note: Hoechst 33342 stock solution is recommended to be stored at -4℃ or -20℃ in the dark after aliquoting. Please avoid repeated freezing and thawing.
2. Preparation of working solution
Dilute the stock solution with pure DMEM or PBS to a final concentration of 10 μg/mL Hoechst 33342 working solution.
Note: Please adjust the concentration of Hoechst 33342 working solution according to actual conditions. It is recommended that you prepare it for immediate use.
2. Cell staining
1. Suspended cells
1) Collect cells by centrifugation and add PBS to wash the cells twice. Wash gently for 5 min each time. The recommended cell density is 1×106/mL
2) Add 1 mL Hoechst 33342 working solution and incubate at room temperature for 3-10 min.
3) Please use a centrifuge, 400 g, centrifuge for 3-4 min, and discard the supernatant.
4) Add PBS to wash the cells twice, gently, 5 min each time.
5) After resuspending the cells with 1 mL of pure DMEM or PBS, observe using a fluorescence microscope or flow cytometer.

2. Adherent cells
1) Culture the adherent cells on a sterile coverslip.
2) Remove the coverslip from the culture medium and aspirate the excess culture medium, gently.
3) Add 100 μL of Hoechst 33342 working solution, gently shake it to completely cover the cells, and incubate at room temperature for 3-10 min.
4) Aspirate the Hoechst 33342 working solution, wash 2-3 times with pure DMEM, 5 min each time, and observe using a fluorescence microscope or flow cytometer.

Notes：
1. You can adjust the working solution concentration of Hoechst 33342 according to your experimental purpose.
2. This product is limited to scientific research and is prohibited from being used for other purposes.
3. For your safety and health, please wear a lab coat and disposable gloves when operating.