DAPI (4',6-Diamidino-2-phenylindole) dihydrochloride is a DAPI dye. DAPI is a fluorescent dye that binds strongly to DNA. It binds to the AT base pair of the double-stranded DNA minor groove, and one DAPI molecule can occupy three base pair positions. The fluorescence intensity of DAPI molecules bound to double-stranded DNA is increased by about 20 times, and it is commonly observed with fluorescence microscopy, and the amount of DNA can be determined based on the intensity of fluorescence. In addition, because DAPI can pass through intact cell membranes, it can be used to stain both live and fixed cells^[1]. DAPI (Compound 3) is an acid-sensing ion channel 3 (ASIC3) inhibitor. DAPI binds to ASIC3 and blocks the channel function. DAPI can be used in the study of chronic pain treatment^[2] (Ex/Em = 356/451 nm).

General Protocol
1. Preparation of DAPI working solution
1.1 Preparation of the stock solution
Dissolve 1 mg DAPI in 1 mL ddH₂O to obtain 1 mg/mL of stock solution.
Note: It is recommended to store the stock solution at -20 °C or -80 °C away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of DAPI working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 ug/mL of working solution.
Note: Please adjust the concentration of DAPI working solution according to the actual situation.
2. Cell staining
2.1 Suspension cells（6-well plate）
a. Centrifuge at 1000 g at 4°C 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°C 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.

Storage
-20°C, 1 year. Protect from light

Precautions 1. Please adjust the concentration of DAPI 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 (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]. Kapuscinski J, et al. DAPI: a DNA-specific fluorescent probe. Biotech Histochem. 1995 Sep;70(5):220-33.  [Content Brief]

  [2]. Kuduk SD, et al. Amidine derived inhibitors of acid-sensing ion channel-3 (ASIC3). Bioorg Med Chem Lett. 2009 Aug 1;19(15):4059-63. Sep;70(5):220-33.  [Content Brief]