Dcfh2 da

Salvia miltiorrhiza Bunge and Radix Puerariae were purchased from Changda Prepared Chinese Medicinal Herbs Co. Ltd (Anguo, China). Healthy SD rats, SPF clean grade, female, 10 weeks old, weight 200–250 g, were purchased from Chinese Academy of Sciences; The ELISA kits for detecting blood urea nitrogen (BUN), creatinine, ALP, OPG and RANKL were purchased from Wuhan Huamei Biological co., LTD (Wuhan, China); DCFH2-DA, Fluo-3/AM, BAPTA-AM and Hoechst 33,258 were obtained from Molecular Probes (Eugene, OR); Chemicals used for DPI, TTRA, AA, ALL, NAC, NDGA, Rot were purchased from Sigma-Aldrich (St. Louis, MO). H&E and Masson dying kits were purchased from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China); 3-methyladenine (3-MA), and monodansyl cadaverine (MDC) were obtained from MedChemExpress; TRAP staining kit was obtained from BIO-SCIENCE COMPANY LIMITED (Shanghai, China); Antibodies GAPDH, LC3B, Beclin-1, p62, p-p65 and NF-κBp65 were purchased from Sigma Company; Antibodies NFATc1 and c-Fos were purchased from Chengdu Zen Bio (Chengdu, China); Secondary antibodies were purchased from American CST Company; Hoechst 33,342 was purchased from Thermo Fisher Scientific; JYB1-1 calcium removal solution and protein extraction kit were purchased from Solarbio Company (Beijing, China); RANKL was purchased from Sino Biological Company (Beijing, China).

Production of intracellular peroxides was performed with non-fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH2-DA). This liposoluble compound enters cells and accumulates at cytosol and is cleaved by intracellular esterases in 2,7-dichlorodihydrofluorescein (DCFH2), which in the presence of peroxides is oxidated to dichlorodihydrofluorescein (DCF), a green fluorescent compound easily visualized with excitation and emission wavelengths of 504 nm and 529 nm, respectively [18 (link)]. Emitted fluorescence and intracellular peroxide concentrations are proportional [20 (link)]. Briefly, a cell suspension of 10⁶ cells was incubated for 45 min, at 37 °C in the dark, with 5 μM of DCFH2-DA (Molecular Probes, Invitrogen^®, Carlsbad, CA, USA). After the cells were washed with PBS, the detection was made by flow cytometry.

Macrophages were pretreated with or without EGCG for 1 h prior to LPS treatment for 24 h. Cells were then washed with serum-free medium and incubated in 10 μM of 2′7′-dichlorofluorescin diacetate (DCFH₂DA; Molecular Probes) at 37°C for 30 min [24 (link)]. After a counterstaining of nuclear with Hoechst 33342 (2 μg/mL) for 5 min and wash, the ROS production was assessed using a fluorescence microscope (Olympus IX71) at 488/527 nm.

Reactive oxygen species (ROS) accumulation in seedlings was detected using the cell-permeable fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA; Molecular Probes) according to Schopher et al. [42] (link). 5-day-old seedlings grown on MS with or without 10 µM ACC were treated with 200 mM NaCl for 6 h. Then, seedling were incubated in 100 µM DCFH2-DA in 1% ethanol for 20 min, and washed with distilled H₂O to remove the dye before the observation of ROS accumulation under the confocal microscope. Confocal images were obtained after excitation at 488 nm and emission at 522 nm. H₂O₂ production was detected in seedlings using 3, 3-diaminobenzidine (DAB) as substrate. Relative electrolyte leakage and chlorophyll content were measured as described previously [60] (link).

The initial rates of lactate production were determined as an index of glycolysis by enzymatic determination of lactate concentrations in the culture medium (38 (link)). Oxygen consumption rates were determined in an XF24 Extracellular Flux Analyzer (Seahorse Bioscience) (22 (link)). Cells were seeded in the microplates, and incubated at 37°C and 5% CO₂ for 24 h. The final concentration and order of injected substances was 6 μM OL, 0.75 mM DNP (2,4-dinitrophenol), 1 μM rotenone, and 1 μM antimycin. The intracellular production of hydrogen peroxide was monitored by flow cytometry using 5 μM 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA) (Molecular Probes, Eugene, OR, USA) and incubated 30 min at 37°C (27 (link)). Cells were analyzed in a BD FACScan. For each analysis, 10,000 events were recorded.

Intracellular total ROS levels were detected by using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA; Molecular Probes, Life Technologies) as described previously [28 (link)]. Cells (10⁵ cells/well) were seeded in a 6-well plate. Serum-deprived cells were treated with and without TGF-β1 (10 ng/ml) for 6 and 24 hrs. Following treatments, trypsinized cells were suspended in Krebs-HEPES buffer containing DCFH₂-DA (10 μM). Fluorescence was then measured with excitation and emission at 490 and 530 nm, respectively, with a Polarstar microplate reader (BMG Labtech) at 37°C.

The ROS generation was assessed using the fluorogenic probe DCFH₂-DA (Molecular Probes, Invitrogen) [19 (link), 28 (link)]. Briefly, injured H9c2 cells, treated with Nar or vehicle, were washed in PBS/10 mM glucose (loading buffer) and loaded with 8 μM DCFH₂-DA for 30 min in the dark (37°C). The cells were then washed and incubated in loading buffer. FDA fluorescence was estimated using a plate reader with wavelengths of 485 nm (excitation) and 520 nm (emission) (Wallac, Victor 2, 1420 multilabel counter, PerkinElmer) 30 minutes later. Then, after washing with PBS/10 mM glucose, the cells were incubated with crystal violet for 30 min at room temperature. After extensive washing, a solution of 1% SDS was added to each well, the plates were mechanically shaken for 1 h, and the absorbance at 595 nm was determined. The DCFH₂-DA fluorescence values were normalized to the cell content of each well as indicated by the crystal violet assay.