N acetylcysteine nac

About 3 × 10⁵ cells were harvested, washed with serum-free medium and incubated with 5 μmol/L dihydroethidium (DHE, Beyotime, Shanghai, China) at 37 °C for 30 min. Then the cells were harvested, washed and resuspended in serum-free culture medium. After staining, wash 2–3 times with PBS, replace the staining solution with fresh culture medium or buffer, and then place it under a fluorescence microscope (excitation 300 nm and emission 610 nm) (OLYMPUS, Tokyo, Japan) for observation and quantification with ImageJ software. Besides, young and senescent MSCs cultured with 10 mM N-Acetyl-cysteine (NAC, a ROS scavenger) (Selleck, Houston, TX, USA) for 24 h to test the specify of DHE [53 (link)].

Antibodies against Drp1, p62, LC3, RIP1, RIP3, TSG101, and CD63 were purchased from Abcam (Cambridge, MA, USA). β-actin, tubulin, and COX IV, which were used as references for the total, cytoplasmic, and mitochondrial fractions, were also acquired from Abcam (Cambridge, MA, USA). Antibodies against Phospho-RIP3 (Thr231/Ser232) and Phospho-Drp1 (Ser616) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Aggresome Detection Kit was acquired from Abcam (Cambridge, MA, USA). MitoTracker Deep Red, the ROS 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit, dihydroethidium (DHE) assay kit, and MitoSOX detection kit were purchased from Invitrogen (Carlsbad, CA, USA). Ad-mRFP-GFP-LC3 was supplied by Beyotime (Shanghai, China). Adenoviral vectors for Drp1 short hairpin RNA (shRNA) and Drp1 mutations (Drp1-S616D and Drp1-S616A) were generated by Genechem Technology (Shanghai, China). Related activators and inhibitors, including Mitochondrial division inhibitor 1 (Mdivi-1), N-acetylcysteine (NAC), and Necrostatin-1 (Nec-1), were obtained from Selleck (Shanghai, China). The Protein A/G Magnetic Beads IP Kit was purchased from Thermo Scientific (Waltham, MA, USA), and fetal bovine serum (FBS) and penicillin/streptomycin were procured by Invitrogen (Carlsbad, CA, USA). All other chemicals were supplied by Sigma unless otherwise specified.

DMEM/F-12, penicillin/streptomycin, amphotericin, and fetal bovine serum (FBS) were from Thermo (Massachusetts, USA); collagenase XI, dispase II, IL-1β, and diacetyl dichlorofluorescein (DCFH-DA) kit were purchased from Sigma (Ohio, United States); Masson's and Safranin O Stain Kit was from Solarbio (Shanghai, China); anti-collagen II (ab34712), anti-PAOX (ab230441), anti-p16 (ab51243), anti-β-actin (ab6276), anti-8-hydroxy-2′-deoxyguanosine (ab48508), anti-gamma H2A.X (ab81299), IgG H&L Alexa Fluor 488 (ab150113), Hydrogen Peroxide Assay Kit (ab138874, ab102500), and Total Polyamine Assay Kit (ab239728) were from Abcam (Cambridge, USA); Trizol reagent and reverse transcriptase kit were from Vazyme (Nanjing, China). SYBR Green Master Mix was from Applied Biosystems (Waltham, USA); Vectastain Elite ABC reagent was obtained from KeyGen (Nanjing, China); spermidine and N-acetylcysteine (NAC) were from Selleck (Shanghai, China); Lipofectamine 2000 transfection reagent (89900) and Protein Extraction Kit (78510) were from Thermo Fisher Scientific (Waltham, USA); ethylenediaminetetraacetic acid (EDTA), paraformaldehyde, and enhanced chemiluminescence (ECL) were from Beyotime (Shanghai, China).