DCFH2-DA (2',7'-Dichlorofluorescein diacetate) (Synonyms: DCFDA, MFCD 37501)

Cell Staining:
This protocol is provided as a guideline only and should be modified according to your specific needs.
1 Preparation of Staining Solution
(1) Preparation of Stock Solution: Dissolve 2',7'-Dichlorofluorescein diacetate (DCFH₂-DA) in DMSO to prepare a stock solution with a concentration of 1–10mM. Aliquot and store in the dark at −20°C to avoid repeated freeze-thaw cycles.
(2) Preparation of Working Solution: Dilute the stock solution with an appropriate buffer (e.g., serum-free culture medium or PBS) to prepare a working solution with a concentration of 1–100μM.
Note: Adjust the working solution concentration according to the actual situation and prepare it freshly when needed.
2 Suspension Cell Staining
(1) Suspension Cells: Centrifuge at 4°C and 1000g for 3–5 minutes, 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.
(3) Resuspend the cells in the DCFH₂-DA working solution and incubate in the dark at room temperature for 5–30 minutes. The optimal incubation time may vary for different cell types; please optimize according to your specific experimental needs.
(4) After incubation, centrifuge at 1000g for 5 minutes, discard the supernatant, and wash 2–3 times with PBS for 5 minutes each time.
(5) Resuspend the cells in pre-warmed serum-free cell culture medium or PBS and observe using a fluorescence microscope or flow cytometry.
3 Adherent Cell Staining
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslips from the culture medium, aspirate excess culture medium, and place the coverslips in a humid environment.
(3) Add 100μL of the staining solution to one corner of the coverslip and gently agitate to ensure even coverage of all cells.
(4) Incubate in the dark at 37°C for 5–30 minutes. The optimal incubation time may vary for different cell types; please optimize according to your specific experimental needs.
(5) After incubation, aspirate the staining solution and wash the coverslip 2–3 times with pre-warmed culture medium.
 4 Microscope Detection: The maximum absorption/emission wavelengths for DCFH₂-DA are 492/515nm.
Notes:
(1) All fluorescent dyes are susceptible to quenching; minimize light exposure to delay the decay of fluorescence.
(2) For your safety and health, wear a laboratory coat and disposable gloves when handling.

References:
[1] Zhao W, Ma G, Chen X. Lipopolysaccharide induced LOX-1 expression via TLR4/MyD88/ROS activated p38MAPK-NF-?B pathway. Vascul Pharmacol. 2014 Dec;63(3):162-72.