2 7 dichlorodihydrofluorescein diacetate dcfh da

Tetraethyl orthosilicate (TEOS), triethanolamine (TEAH), coumarin (C₉H₆O₂, 99%), 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA), sodium dithionite (SDT) and decahydronaphthalene (98%) were purchased from Sigma-Aldrich. Potassium permanganate (97%) was obtained from Shanghai Chemical Co. Dichloromethane (99.99%) was purchased from Aladdin Industrial Inc. Dodecacarbonyltriiron (Fe₃(CO)₁₂, 96%) was purchased from Macklin Industrial Inc. All chemicals were used as received without any further purification. Hemoglobin (Hb) of bovine red blood cells was purchased from MP Biomedicals. DAPI, Lyso-Tracker Green, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and 2-[6-(4,-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) were purchased from Beyotime Biotechnology.

Purified Xn (# 6754-58-1, HPLC analysis ≥99%, C₂₁H₂₂O₅, MW: 354.40) was from Bide Pharmaceutical Technology Co., Ltd. (# BD156629, Shanghai, China) (chemical structure shown in Figure 1A). NaT (# 86339), Collagenase IV (# C4-BIOC), Triton X-100 (# X100) were from Sigma-Aldrich (Saint Louis, United States). Hoechst 33342 (# 62249) and propidium iodide (PI; # P1304MP) were from Molecular Probes (Eugene, United States). Lactase dehydrogenase (LDH)-Glo™ Cytotoxicity Assay Kit (# J2380) was provided by Promega Corporation (Madison, United States). Protease and phosphatase inhibitors (# P1045), RIPA (# P0013C), IL-6 ELISA kit (# PI326), IL-10 ELISA kit (# PI522), IL-17 ELISA kit (# PI545), TNF-α ELISA kit (#PT512), malondialdehyde (MDA) activity assay kit (# S0131S), superoxide dismutase (SOD) activity assay kit (# S0103) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; # S0033S) were all from Beyotime Biotechnology (Shanghai, China). The following antibodies were used: Nrf2 (# bs-1074R), p-P65(# bs-3543R), P65(# bs-23217R) were from Bioss (Beijing, China); HO-1 (# 43966), p-AKT (# 5012), p-mTOR (# 5536), LC3B (# 3836), β-actin (# 58169), GAPDH (# 5174) were from Cell Signaling Technology (Danvers, United States). P62 (# A11483) was from ABclonal Technology (Wuhan, China). SC79 (# HY-18749) and MHY1485 (# HY-B0795) were from MedChemExpress (South Brunswick, United States).

FeCl₃•6H₂O 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), and MB were procured from J&K Scientific Co., Ltd (Beijing, China). Polyvinylpyrrolidone (PVP, MW =8000) was purchased from Titan Scientific Co., Ltd (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). The Calcein-AM/PI double staining kit and 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) were procured from Beyotime Biotechnology Co., Ltd. (Beijing, China). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was obtained from MedChemExpress (NJ, USA). Cur, Indocyanine Green (ICG), 1-methyl-4-phenylpyridinium ion (MPP⁺), and Levodopa (L-DOPA) were obtained from Sigma-Aldrich (St. Louis, USA). Reactive oxygen species (ROS), adenosine triphosphate (ATP), and malondialdehyde (MDA) ELISA kits were purchased from R&D Systems, Inc., USA.

Anlotinib was kindly provided by Chia Tai Tian Qing Pharmaceutical Group Co., Ltd (Nanjing, China). Metformin, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), methanol, crystal violet, and phosphate-buffered saline (PBS) were purchased from Solarbio Bioscience & Technology Co. Ltd (Beijing, China). Hoechst 33342, propidium iodide (PI), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and SDS lysis buffer were obtained from Beyotime Biotechnology Co., Ltd (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin, and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA, United States).

2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Biotechnology, China), a permeable and sensitive probe, was used for the detection of ROS in this study. We randomly selected 10 embryos at 48 hpf and stained them for 40 min with 100 μM of DCFH-DA diluted in PBS. After this was washed out with PBS for 10 min, all groups were photographed under a fluorescence microscope under a fluorescein isothiocyanate channel. All steps were finished in the absence of light.

Tetramethyl rhodamine, methyl ester (TMRM) staining can quantify changes in mitochondrial membrane potential in living cells. It was used to monitor mitochondrial function. Cells were incubated with TMRM (Sigma, Steinheim, Germany) staining solution at 37°C for 20 min in the dark. Then TMRM was replaced with Hank’s solution, and finally, cells were viewed and photographed by ImageXpress Microsystem High-content imaging system (Molecular Devices).
Intracellular reactive oxygen species (ROS) generation level was determined by using the probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime Biotechnology, Shanghai, China). Briefly, cells were incubated with DCFH-DA in DMEM for 20 min. After washing twice with PBS, cells were observed and photographed under a fluorescence microscope (Olympus IX71, Tokyo, Japan).

ROS measurement was carried out by kit as described previously [26 (link)]. For intracellular ROS staining, individual cells were inoculated in 6-well plate and treated with interfering strands, plasmids, or NAC (5 mM, 4 h) (Beyotime, Shanghai, China) and tempol (3 mM, 4 h) (MCE, New Jersey, USA), and then, 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime) or 10 μM DHE (Beyotime) was used to stain the cells at 37°C for 30 min. The immunofluorescence image was taken with Olympus DP70 digital camera and Olympus BX51 microscope (Olympus, Tokyo, Japan). Green fluorescence (DCF) intensity was quantitated using the microscope with 488 nm excitation and 525 nm emission settings, respectively. Red fluorescence (HE) was measured with 300 nm excitation and 610 nm emission settings.