Reactive oxygen species assay kit

Myr was purchased from Jingzhu Pharmaceutical Co., Ltd. (529-44-2). The CCK-8 assay kit (96992-100TESTS-F) was purchased from Sigma. The BCA assay kit (P0011) and Hoechst 33258 (C1017) were purchased from Beyotime Institute of Biotechnology. Transwell chambers (3422, BD), artificial basement membrane (356234, BD), 0.5% crystal violet (60506ES60), and reactive oxygen species assay kit (50101ES01) were purchased from Yeasen Biotech Co., Ltd. LDH (lactate dehydrogenase, A020-2), MDA (malondialdehyde, A003-1) and SOD (superoxide dismutase, A001-3) detection kits were purchased from Nanjing Jiancheng Institute of Biotechnology.

Recombinant rat fractalkine (E. coli‐derived chemokine domain of rat fractalkine protein, Gln25‐Gly100) was purchased from R&D (537‐FT‐025/CF, Shanghai, China). Streptozotocin (STZ, S0130) and glyoxal (50649) were purchased from Sigma‐Aldrich. The cell counting kit (CCK‐8, 40203ES60), protein extraction RIPA lysis buffer (PC101), protease inhibitor cocktail (20123ES10), phosphatase inhibitor cocktail (20109ES05), SYBR green real‐time PCR master mix (11123ES60) and reactive oxygen species assay kit (50101ES01) were purchased from Shanghai Yeasen Biotechnology Co. Ltd. DMEM (High Glucose, sh30243.01) and DMEM (Low Glucose, SH30021.01) were purchased from HyClone. Foetal bovine serum was purchased from Gibco (10091148, USA). Penicillin/streptomycin (15140155) was purchased from Invitrogen. Pierce BCA protein assay kit (23225) was purchased from Thermo Scientific. The information of the antibodies, used for Western blot and immunofluorescence, was provided in Table 1.

Quercetin (Q4951, purity > 95%, Sigma-Aldrich), erastin (HY-15763, purity > 99% MedChemExpress) and ferrostatin-1 (SML0583, purity > 95%, Sigma-Aldrich) were dissolved in DMSO. For the cell experiments, Quercetin was dissolved in DMSO to achieve the pre-designed concentration and cells were treated with Quercetin for 24 h or 48 h. The following kits were used in the present study: Cell-Counting-Kit-8 (CCK-8) kit, DAPI Staining Solution, Crystal Violet Staining Solution, Calcein/PI Live/Dead Viability/Cytotoxicity Assay kit, Cell Cycle and Apoptosis Analysis kit, Cell Cycle and Apoptosis Analysis kit, mitochondrial membrane potential assay kit with JC-1 (Beyotime, China); Reactive Oxygen Species Assay kit (YEASEN, China). The antibodies used in this study included primary antibodies to anti-p53, anti-xCT, anti-glutathione peroxidase 4, anti-aconitase 1 (ACO1), anti-transferrin receptor, anti-ferritin light chain and anti-collagen IV (Abcam, USA); GAPDH (D16H11) XP rabbit mAb, anti-rabbit IgG, horse radish peroxidase (HRP)-linked antibody, anti-mouse IgG, and HRP-linked antibody (Cell Signaling Technology, USA).

The intracellular ROS production was measured by Reactive Oxygen Species Assay Kit (Yeasen, Shanghai, China), which is a change in fluorescence intensity based on fluorescent dye DCFH-DA (2, 7 - dichlorodi - hydrocein diacetate). Three vials of HFF cells infected with RH strain were added with 5 μM compounds and DMSO, respectively. Extracellular parasites were collected according to the above methods, washed with PBS buffer solution until the final concentration was 1×10⁶ parasites/ml, and then mixed with 10 μM DCFH-DA. The mixture was incubated at 37°C in the dark for 30min, mixed upside down every 5 min, and detected directly on flow cytometry with 3 replicates.

The intercellular ROS was determined by Reactive Oxygen Species Assay Kit (Yeason, ShangHai, China). We used DCFH-DA (1:1000) diluted in M2 medium to incubate the different groups of living oocytes for 30 min. The Annexin V-FITC Apoptosis Detection kit was used to detect apoptosis. The living oocytes washed with PBS were incubated for 30 min in Annexin V-FITC combination buffer (195 μL) added with Annexin V-FITC (5 μL). Finally, the staining of oocytes were captured with a Zeiss Axio Observer D1 microscope (Carl Zeiss, Inc., Thornwood, NY). The procedure of staining and settings of microscope were keeping same for each group.

GSH was detected by a GSH and GSSG Assay Kit (Beyotime, S0053). Cells were washed with PBS. Fresh cells were collected, the corresponding reagents were added according to the reagent instructions and centrifuged, the corresponding working detection solution was added (blank and standard control were set), the absorbance value of A412 was detected on the microplate reader, and the results were processed in GraphPad Prism 8 software. ROS were detected by a Reactive Oxygen Species Assay Kit (YEASEN, 50101ES01). When the cells were 50 to 70% confluent, the medium was removed, and the fluorescent dye probe working solution (2,7-dichlorodihydrofluorescein diacetate, DCFH-DA) was added (negative control was set). The cells were incubated at 37°C in the dark for 30 min. The treated cells were examined by flow cytometry, and the results were processed in FlowJo_V10 software.