H2dcf

AA, dithiothreitol (DTT), H2DCF, H₂O₂, DMPO, DMS, DMSO, and penicillin–streptomycin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). MitoSox red was purchased from Invitrogen (Waltham MA, USA). DMEM/F12, RPMI1640 and fetal bovine serum were purchased from HyClone (Logan, UT, USA). NaBH₄ was obtained from Sinopharm chemical reagent (Shanghai, China). SPME holder and 75‐μm carboxen‐PDMS SPME fiber was purchased from Supelco (Bellefonte, PA, USA). Ni‐Trap nickel‐chelating column was obtained from Qiagen (Hilden, Germany). Thrombin kit was purchased from New England Biolabs (Ipswich, MA, USA). Fast Mutagenesis System was obtained from Transgene (Beijing, China). MDA and cell survival rate kits were obtained from Jiancheng Biotech (Nanjing, China).

To determine the intracellular ROS levels, cells were stained with 2′,7′-dichlorodihydrofluorescein acetate (H2-DCF; Sigma-Aldrich), and fluorescence intensity was measured according to the fluorescence detection conditions of FITC using the MACSQuant Analyzer (Miltenyi Biotech). A membrane potential probe, the 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)), was used to evaluate the mitochondrial membrane potential. Cells were incubated with 10 μM DiOC2(3) (Thermo Fisher Scientific, Milan, Italy) for 30 min at 37 °C, washed twice, resuspended in PBS and analyzed by flow cytometry through the detection of the green fluorescence intensity of DiOC2(3).

Reactive oxygen species (ROS) were detected using 2′,7′-dichlorodihydrofluorescein acetate (H2-DCF; Sigma-Aldrich, St. Louis, MO, USA), and fluorescence intensity was measured according to the fluorescence detection conditions of FITC by using a MACSQuant Analyzer (Miltenyi Biotech, North Rhine-Westphalia, Germany), as already reported [21 (link)].
Moreover, to measure changes in the mitochondrial mass, cells were reacted with 200 nM MitoTracker Red CMXRos probe (Thermo Fisher Scientific, Milan, Italy) for 30 min at 37 °C, according to the manufacturer′s instructions. After being washed twice, cells were treated. Then, 24 h later, they were analyzed by flow cytometry.