H2DCFDA (DCFH-DA) is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm)^[1].

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
1. Prepare the storage solution and working solution 1.1 Prepare a 10 mM stock solution of H2DCFDA using anhydrous DMSO.
1.2 Dilute the stored solution with pre-warmed serum-free cell culture medium or PBS to prepare a working solution of 1-10 μM H2DCFDA.
Note: Please adjust the concentration of the H2DCFDA working solution according to the actual situation, and prepare it as needed.
2. Cell Staining (Suspension Cells) 2.1 Centrifuge the cells and add PBS for two washes, each for 5 minutes. The cell density is 1×10⁶ cells/mL.
2.2 Add 1 mL of the dye working solution and incubate at room temperature for 5 to 30 minutes.
2.3 400 grams, centrifuge for 3-4 minutes, then 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. Cell Staining (Monolayer Cells) 3.1 Cultivate adherent cells on sterile coverslips.
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 5 to 30 minutes.
3.4 Remove the dye working solution and wash with the culture medium 2-3 times, for 5 minutes each time. Then observe using a fluorescence microscope or flow cytometer.
Note: If flow cytometry is to be used for detection, the cells should first be digested with trypsin, resuspended, and then stained.

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 (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Lyublinskaya OG, et al. Redox environment in stem and differentiated cells: A quantitative approach. Redox Biol. 2017 Aug;12:758-769.  [Content Brief]