To investigate whether H2O2 induces oxidative stress in GCs, ROS detection was performed using a reactive oxygen species assay kit (KeyGen, Shanghai, China). Briefly, GCs were incubated with DCFH-DA (diluted at 1:1,000 in serum-free medium) at 37 °C for 20 min. Cells without DCFH-DA treatment were used as negative controls. After washing twice with serum-free medium, the fluorescence of the DCF in the cells was detected at an excitation wavelength of 488 nm with a BD FACScan flow cytometer (Becton Dickinson, Franklin, NJ, USA).
Reactive oxygen species assay kit
Manufactured by Keygen Biotech
Sourced in China
The Reactive Oxygen Species Assay Kit is a laboratory tool designed to quantify the levels of reactive oxygen species (ROS) in biological samples. It provides a standardized and reliable method for measuring various ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals. The kit includes all the necessary reagents and protocols to perform the assay, enabling researchers to assess oxidative stress levels in their experiments.
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22 protocols using reactive oxygen species assay kit
1
Measuring Oxidative Stress in Granulosa Cells
2
Measurement of ROS
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Exponentially growing cells were treated with DIM (0, 20, 30 lM) for 2 h before exposure to 10 Gy c-irradiation, followed by harvesting at 2 h after irradiation. Intracellular ROS levels were measured using the Reactive Oxygen Species Assay Kit (KeyGEN BioTECH, Nanjing, China) . The fluorescence intensity was proportional to the level of cellular ROS. After the indicated treatment, cells were harvested and performed according to the manufacturer's instructions. The fluorescence of the cells was monitored using flow cytometry (FACSCalibur, BD). ROS production was calculated as the fold increase in the fluorescence compared with that observed in the control.
3
Evaluating Manganese-Induced Oxidative Stress
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The logarithmically growing cells were inoculated into 6-well plates with a quantity of 3×105/well and treated with 0, 125, 250, and 500 μM MnCl2 for 6 h. Subsequently, ROS levels were detected using the Reactive Oxygen Species Assay Kit (KeyGEN BioTECH). DCFH-DA was diluted with serum-free DMEM at 1:1000 to reach a final concentration of 10 μmol/L. After removing the medium, cells were washed 3 times with PBS, and 1 mL diluted DCFH-DA probe was added. Following incubation for 20 min at 37°C, the cells were washed with PBS to fully remove DCFH-DA. The fluorescence intensity of 10,000 cells was detected by flow cytometry under the FITC channel. Simultaneously, changes in mitochondrial membrane potential were detected with the JC-10 Apoptosis Detection Kit (KeyGEN BioTECH). The treated cells were collected by trypsinization and then labeled with JC-10 for 30 min at 37°C. Polarized mitochondria in the aggregate form and depolarized mitochondria in the monomeric form were stained with green and orange fluorescence, respectively. Thus, green and orange emission light were analyzed by fluorescence channel 1 (FL1) and fluorescence channel 2 (FL2) using flow cytometry, respectively.
4
ROS detection
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The ROS detection was performed by Reactive Oxygen Species Assay Kit (KeyGen, Shanghai, China). Brie y, GCs were incubated with DCFH-DA (diluted at 1:1000 in serum-free medium ) for 20 min in 37 ℃, and the uorescence of DCF in cells was detected by using BD FACScan ow cytometry (Becton Dickinson, Franklin, NJ, USA).
5
Mitochondrial Stress Assay in Cells
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BaP was purchased from Sigma (St. Louis, MO, USA). S9 Fractions (S9) was purchased from Gibco (ThermoFisher Scientific, Shanghai, China). BHA was purchased from Sangon Biotech, Shanghai, China. The Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), oligomycin, rotenone antimycin A, and 2-deoxyglucose (2-DG) were purchased from Sigma Aldrich, Shanghai, China. MEM / F12 medium and fetal bovine serum (FBS) were purchased from Procell Life Science & Technology, Wuhan, China. Trypsin-EDTA solution, penicillin / streptomycin mixture, and dimethyl sulfoxide (DMSO) were purchased from Solarbio Life Sciences, Beijing, China. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation and cytotoxicity assay kit, Malondialdehyde (MDA) assay kit, and Superoxide Dismutase (SOD) typed assay kit were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Annexin V -FITC Apoptosis Detection Kit, Reactive Oxygen Species Assay Kit, and JC-1 Apoptosis Detection Kit were purchased from Jiangsu KeyGEN BioTECH, Nanjing, China.
6
Intracellular ROS Measurement After DIM+IR
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Exponentially growing cells were treated with DIM (20 μM) for 2 h before exposure to 10 Gy γ-irradiation, followed by harvesting at 2 h after irradiation. Intracellular ROS levels were measured using the Reactive Oxygen Species Assay Kit (KeyGEN BioTECH, Nanjing, China). The fluorescence intensity was proportional to the level of cellular ROS. After the indicated treatment, cells were harvested and performed according to the manufacturer's instructions. The fluorescence of the cells was monitored using flow cytometry (FACSCalibur, BD, Franklin Lakes, New Jersey, USA). ROS production was calculated as the fold increase in the fluorescence compared with that observed in the control.
7
Intracellular ROS Evaluation of BP Nanomaterials
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The effect of BP nanomaterials and corona complexes on intracellular ROS generation in macrophage cells was detected with a Reactive Oxygen Species Assay Kit (KeyGEN BioTECH Co., Ltd, Nanjing, China) following the manufacturer’s protocol. Briefly, dTHP-1 cells were seeded onto 96-well plates at 1.0 × 106 cells per well for 24 h, and then the culture medium was replaced with 100 μl of DCF-DA/DMEM44 medium (final DCF concentration is 10 μM) and incubated at 37 °C for 20 min. Then BP nanomaterials (100 μg ml−1) and corona complexes (100 μg ml−1) were added to the wells. The fluorescence intensity of DCF was measured by microplate reader (Tecan Infinite M1000 PRO) with the excitation and emission wavelengths at 488 and 525 nm, respectively, and the fluorescence intensity was used to evaluate the ROS level in macrophage cells. All experiments were conducted at least twice to ensure the reproducibility of the results.
8
Reactive oxygen species (ROS) detection
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L02 cells and liver samples were analysed for ROS content by using a Reactive Oxygen Species Assay Kit (KeyGen BioTECH, KGT010, Nanjing, China). Briefly, 1 × 106 cells, and 1 g liver samples were collected, rinsed with cleaning liquid from the kit, and then centrifuged at 300 × g for 5 min. ROS content of the precipitations was then measured by fluorescence spectrophotometry according to the manufacturer's instructions.
9
ROS Measurement in Treated Cells
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Reactive Oxygen Species Assay Kit (KeyGEN BioTECH, Nanjing, China) was used to detect intracellular ROS levels. Exponentially growing cells were treated with UA (4μM), Adr (4μM), and UA plus Adr (4μM, respectively) and incubated for 48h before harvesting and performed according to the manufacturer's instructions. The fluorescence of the cells was monitored using flow cytometry (FACSCalibur, BD, Franklin Lakes, New Jersey, USA). ROS production was calculated as the intensity in the fluorescence compared with the control group.
10
Detection of reactive oxygen species (ROS)
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Reactive Oxygen Species Assay Kit (KeyGen Biotech, Nanjing, Jiangsu) was used to
measure the levels of ROS in SRA01/04 cells following the protocol of the
manufacturer. SRA01/04 cells (2 × 105 cells/well) were incubated with
200 μM H2O2 in a 6-well plate for 24 h. After that,
SRA01/04 cells were stained with 10 μM DCFH-DA at darkness for 25 min. The
fluorescence intensity of the SRA01/04 cells was observed under a confocal laser
scanning microscope.
measure the levels of ROS in SRA01/04 cells following the protocol of the
manufacturer. SRA01/04 cells (2 × 105 cells/well) were incubated with
200 μM H2O2 in a 6-well plate for 24 h. After that,
SRA01/04 cells were stained with 10 μM DCFH-DA at darkness for 25 min. The
fluorescence intensity of the SRA01/04 cells was observed under a confocal laser
scanning microscope.
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