Hoechst 33342 trihydrochloride 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].

Dye reagent^[1]
DNA Stain^[1]

Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.)
1. Preparation of Hoechst working solution
1.1 Preparation of storage solution
Prepare a 1 mg/mL storage solution of Hoechst using DMSO.
1.2 Preparation of working solution
Dilute the stored solution with pre-heated serum-free cell culture medium or PBS to achieve a final concentration of 10 μg/mL Hoechst working solution.
Note: Please adjust the concentration of Hoechst working solution according to the actual situation, and prepare it as needed.
2. Cell Staining (Suspension Cells)
2.1 Centrifuge to collect the cells, then add PBS for two washes, each for 5 minutes. The cell density is 1×10⁶/mL
2.2 Add 1 mL of Hoechst working solution and incubate at room temperature for 3-10 minutes.
2.3 400 grams. Centrifuge for 3-4 minutes. Discard the supernatant.
2.4 Add PBS to wash the cells twice, for 5 minutes each time.
2.5 After resuspending the cells in 1 mL of serum-free medium or PBS, observe them using a fluorescence microscope or a flow cytometer.
3. Cytological staining (adherent cells)
3.1 Place the adherent cells on a sterile cover slip.
3.2 Remove the cover glass from the culture medium and aspirate away the excess medium.
3.3 Add 100 μL of the dye working solution and gently shake to ensure complete coverage of the cells. Incubate for 3 to 10 minutes.
3.4 Remove the dye working solution and wash with the culture medium 2-3 times, for 5 minutes each time. Observe using a fluorescence microscope or flow cytometer.

Notes:
1. Please adjust the concentration of Hoechst working solution according to the actual situation.
2. This product is exclusively for professional scientific research purposes only. It shall not be used for clinical diagnosis or treatment, nor for food or medicine purposes.
3. For your safety and health, please wear a laboratory coat and put on disposable gloves when operating.

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 (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]. Chazotte B. Labeling nuclear DNA with hoechst 33342. Cold Spring Harb Protoc. 2011 Jan 1;2011(1):pdb.prot5557.  [Content Brief]