DAPI (hydrochloride) (Synonyms: 4',6Diamidino2phenylindole, FxCycle Violet)

This plan only provides a guide, please modify it to meet your specific needs.
1. Preparation of staining solution
(1) Prepare dye stock solution: Use ddH2O to dissolve DAPI and prepare a stock solution with a concentration of 1-10 mg/ml.
Notice:
① Do not use buffer to dissolve the powder directly;
② The prepared stock solution needs to be filtered and sterilized using a 0.22μm filter membrane;
③ Unused stock solution should be aliquoted according to single dosage and stored in the dark at -20℃ or -80℃ to avoid repeated freezing and thawing.
(2) Prepare dye working solution: Use ddH2O or PBS to dilute the stock solution and prepare a working solution with a concentration of 0.5-10μg/ml.
Note: Please adjust the concentration of the working fluid according to the actual situation and prepare it now.

2. Cell suspension staining
(1) Suspension cells: Centrifuge at 1000g for 3-5 minutes at 4℃, discard the supernatant, and wash twice with PBS for 5 minutes each time.
(2) Adherent cells: Wash twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.
(3) Add 1 mL of DAPI working solution to resuspend the cells and incubate at room temperature in the dark for 5-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.
(5) Resuspend the cells in pre-warmed serum-free cell culture medium or PBS. Observe by fluorescence microscopy or flow cytometry.
3. Cell adhesion staining
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100 μL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.
(4) Incubate at room temperature in the dark for 3-5 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(5) After the incubation, discard the dye working solution and use pre-warmed culture solution to wash the coverslip 2 to 3 times.
4. Microscope detection: The maximum excitation/emission light of DAPI is 364/454nm respectively.

Precautions:
① DAPI is suitable for staining fixed cells or tissues, usually after other staining;
② To slow down fluorescence quenching, anti-fluorescence quenching mounting agent can be used;
③ Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;
④ DAPI is irritating to the human body. For your safety and health, please wear a lab coat and disposable gloves when operating.