H2DCFDA

H2DCFDA (DCFH-DA) is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm)^[1]. In Vitro:Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1.1 Preparation of stock solution
Use anhydrous DMSO to prepare a 10 mM H2DCFDA stock solution.
Note: It is recommended that the H2DCFDA stock solution be stored at -20 ℃ or -80 ℃ in the dark after aliquoting.
1.2 Preparation of working solution
Use preheated serum-free cell culture medium or PBS to dilute the stock solution to prepare a 1-10 μM H2DCFDA working solution.
Note: Please adjust the concentration of H2DCFDA working solution according to actual conditions and prepare it before use.
2. Cell staining (suspended cells)
2.1 Collect cells by centrifugation and wash twice with PBS for 5 minutes each time. The cell density is 1×10⁶/mL.
2.2 Add 1 mL of dye working solution and incubate at room temperature for 5-30 minutes.
2.3 Centrifuge at 400 g for 3-4 minutes and discard the supernatant.
2.4 Add PBS to wash the cells twice, 5 minutes each time.
2.5 Resuspend the cells in 1 mL of serum-free medium or PBS and observe using a fluorescence microscope or flow cytometer.
3. Cell staining (adherent cells)
3.1 Culture the adherent cells on a sterile coverslip.
3.2 Remove the coverslip from the culture medium and remove the excess culture medium.
3.3 Add 100 μL of dye working solution, gently shake to completely cover the cells, and incubate for 5-30 minutes.
3.4 Aspirate the dye working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope or flow cytometer.
Note: If flow cytometry is required, the cells need to be digested with trypsin and resuspended before staining.