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).

Guidelines (The following is our recommended protocol. This protocol only provides guidance and should be modified according to your specific needs).
1. Preparation of DAPI dihydrochloride working solution
Dilute the stock solution with serum-free cell culture medium or PBS to obtain a working solution of 1-10 μg/mL.
Note: Please adjust the concentration of the DAPI dihydrochloride working solution according to your needs.
2. Cell staining
2.1 Suspension cells (96-well plate)
a. Centrifuge at 1000 g at 4°C for 3-5 minutes, then discard the supernatant. Wash twice with PBS for 5 minutes each time. Adjust the cell density to 1×10⁶/mL.
b. Add 1 mL working solution and incubate at room temperature for 3-10 minutes.
c. Centrifuge at 400 g at 4°C for 3-4 minutes and discard the supernatant.
d. Wash twice with PBS for 5 minutes each time.
e. Resuspend the cells in serum-free cell culture medium or PBS. Observe using a fluorescence microscope or flow cytometry.
2.2 Adherent cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslips from the culture medium and aspirate excess medium.
c. Add 100 μL working solution, gently shake to fully cover the cells, and incubate at room temperature for 3-10 minutes.
d. Wash twice with culture medium for 5 minutes each time. Observe using a fluorescence microscope or flow cytometer.

Storage
Store at -20°C for 1 year. Protect from light.

Notes 1. Please adjust the concentration of the DAPI dihydrochloride working solution according to actual conditions.
2. This product is for R&D use only and is not intended for use in drugs, household, or other purposes.
3. For your safety and health, please wear a lab coat and disposable gloves during operation.

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]