2 7 dichlorodihydrofluorescein diacetate h2dcfda

Cells were seeded into a 6-well plate at a density of 4×10⁵ cells/well, and incubated with different concentrations of UP302 (30, 60, 90, and 120 μM) for 16 h, and dye according to the instructions for use. All samples were subject to FCM detection. All data were quantified using FlowJo 10.1.
Cell apoptosis was quantified with double staining for fluorescein isothiocyanate-conjugated Annexin-V and propidium iodide (Biouniquer, China). Treated cells were collected and washed with PBS, resuspended in 100μL 1x binding buffer, and stained with 5μL annexin V-FITC (AV) and 5 μL propidium iodide (PI) for 20 min at 37 ℃ in the dark. Subsequently, 400μL of 1x binding buffer was added.
ROS levels were determined using 2',7'-dichloro-dihydro fluorescein diacetate (H2-DCFDA, Beyotime Biotechnology, China). Propidium iodide was used to assess the cell cycle. MMP was measured using a JC-1 probe. Assays were performed in triplicate and repeated thrice independently.

Iso (purity ≥ 98%) was obtained from Pharmaceutical Technology Co., Ltd. (Jiangsu, China) and BaP was purchased from Sigma (St. Louis, MO, USA). Caspase-1 activity assay kit and 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) were obtained from Beyotime biotechnology Co., Ltd. (Shanghai, China). RPMI-1640 medium, BCA protein kit, and penicillin–streptomycin solution were from Thermo Fisher (Shanghai, China).
TritonX-100, DAPI-Fluoromount-G, Bovine Serum Albumin (BSA), and CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) were from Proteintech Group, Inc (Rosemont, IL, USA).
Polyclonal antibodies specific to NLRP3 (GTX106313), ASC (10500-1-AP), NF-κΒ (10745-1-AP), GSDMD (20770-1-AP), IL-1β (16806-1-AP) and IL-18 (SC-6179) were purchased from Santa Cruz Biotechnology (Dallas, Texas, United States). GAPDH (BA2913) was obtained from Bioworld Technology, Inc. (Louis Park, MN, USA). Caspase-1 (2225) was purchased from Cell Signaling Technology (Shanghai, China).
Specific inhibitors MCC950, N-acetylcysteine (NAC), and Z-YVAD-FMK were obtained from Beyotime biotechnology Co., Ltd. (Shanghai, China). Ammonium pyrrolidinedithiocarbamate (PDTC) and Mito-TEMPO were obtained from Sigma (St. Louis, MO, USA). All other reagents were dissolved in water or DMSO, and then diluted with fresh RPMI-1640 medium.

The 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) (Beyotime, Jiangsu, China) and Cell Tracker Blue CMF2HC molecular probes (Invitrogen Inc, Carlsbad, CA, USA) were used to detect intracellular ROS and GSH levels, respectively. GV-stage oocytes were collected from bovine ovary and divided into three groups (IOs, IOs with 10⁻⁹ M melatonin-treated, COCs control) and cultured for 23 h in maturation medium. Then, MII-stage oocytes were collected and denuded from adherence of cumulus cells, then they were incubated (in the dark) for 30 min in M2 medium containing H2DCFDA (10 μM) or Cell Tracker Blue (10 μM), respectively. The MII-stage oocytes were collected and denuded from the adherence of cumulus cells, and placed in 30 μL M2 droplets, and then the fluorescence was observed using an epifluorescence microscope. The fluorescence intensity was analyzed using ImageJ software (version 1.40; National Institutes of Health, Bethesda, MD, USA).