H2DCFDA (DCFH-DA) (Synonyms: DCFH, DCFHDA)

This plan only provides a guide, please modify it to meet your specific needs.
1. Preparation of staining solution
(1) Prepare stock solution: Dissolve H2DCFDA (DCFH-DA) in DMSO and prepare a stock solution with a concentration of 1-10mM.
Note: Unused stock solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium or PBS) and prepare a working solution with a concentration of 1-10μM.
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°C, 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 H2DCFDA (DCFH-DA) working solution to resuspend the cells, and incubate at 37 °C 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) Incubate cells with pre-warmed serum-free cell culture medium or PBS.
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 37 °C 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.
(5) After the incubation, discard the dye working solution and use pre-warmed culture solution to wash the coverslip 2 to 3 times.
(6) Incubate cells with pre-warmed serum-free cell culture medium or PBS.
4. Microscope detection: The maximum excitation/emission wavelength of H2DCFDA (DCFH-DA) is 488/525nm.

Precautions:
① For diacetate derivatives, a short recovery time is required for intracellular esterase to hydrolyze them, allowing the dye to respond to oxidative stress. There is a wide range of optimal recovery times, as certain cell types often display extremely low levels of esterase activity;
② Before exposing cells to experimental stimuli, the background fluorescence intensity of cells needs to be measured;
③ For flow cytometry analysis, the forward angle scattering and side angle scattering of cells are unchanged before and after dye treatment, and changes in cell size may be caused by drug treatment or toxic reactions;
④ It is recommended to perform fluorescence detection on experimental systems that do not contain cells. In the absence of extracellular esterases and other oxidases, the gradual increase in fluorescence over time may be related to factors such as instantaneous hydrolysis, air oxidation, and light-induced oxidation;
⑤ For control group (unmedicated) cells, intracellular enzymes or natural antioxidants will scavenge oxygen free radicals. After the dye loading recovery time, healthy cells should show a low-level fluorescence signal and be relatively stable during the entire experiment;
⑥ A gradual increase (due to autoxidation) or a decrease (due to intracellular dye loss or photoquenching) of the fluorescence signal may be observed;
⑦ In a system that does not contain any irritants or inducers, strong fluorescence is suddenly found in healthy and untreated cells, indicating cell death or some other oxidative events;
⑧ Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;
⑨ For your safety and health, please wear a lab coat and disposable gloves.