PC12 cells were seeded at a concentration of 8×104 cells/well in 6-well plates (n = 3 parallel wells per group) for 24 h. The cells were randomly divided into the NC, M, and FLX groups and the eight iridoid groups (CAT/GE/GEN/GPA/AU/AJU/RC/RD, 10 µM) obtained by MTT for activity. After 24 h of drug treatment, the cells were trypsinized and collected by centrifugation. The proportion of apoptotic cells was analyzed by flow cytometry according to the Annexin V-FITC/7-AAD apoptosis kit (Elabscience, Wuhan, China) manual. The ROS levels were determined by flow cytometry according to the reactive oxygen species assay kit (Solarbio, Beijing, China) manual. The evaluation of MMP was determined by flow cytometry according to the Mitochondrial Membrane Potential assay kit with the JC-1 (Beyotime, Shanghai, China) manual.
Reactive oxygen species assay kit
Manufactured by Solarbio
Sourced in China, United States
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 straightforward method for measuring various ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Dspe mpeg2000, by Xi’an Ruixi (3 mentions)
Catalase cat activity assay kit, by Solarbio (2 mentions)
Superoxide dismutase sod activity assay kit, by Solarbio (2 mentions)
Lipid peroxidation mda assay kit, by Beyotime (2 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Solarbio (2 mentions)
Confocal laser scanning microscope, by Leica (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Solarbio (2 mentions)
Cytexpert 2, by Beckman Coulter (2 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Solarbio (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Bca assay kit, by CoWin Biotech (2 mentions)
Mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Iron assay kit, by Solarbio (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Mda test kit, by Solarbio (1 mentions)
74 protocols using reactive oxygen species assay kit
1
Apoptosis and ROS Modulation in PC12 Cells
2
Measuring Intracellular ROS in Monocytes
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The Reactive Oxygen Species assay kit (CA1410-100 T, Solarbio, China) was used to measure the intracellular production of ROS. Fluorescence-free dichloro-dihydro-fluorescein diacetate (DCFH-DA) reagent (final concentration 10 μmol/L) was diluted in a serum-free medium at a ratio of 1:1000. Bone marrow-derived monocytes were treated with different concentrations of anagliptin for 24 h and then were collected and suspended in DCFH-DA. The cells were then incubated for 30 min in a 37 ℃ cell culture incubator. The cell suspensions were mixed every 5 min so that the probe was in full contact with the cells. After incubation, the cells were washed with serum-free cell culture, and M-CSF (20 ng/mL) was added at different time points. The percentage of DCFDA-positive cells was quantified using flow cytometry.
3
Quantifying ROS in MCF-7 Cells
4
Ferroptosis Regulation in Ovarian Cancer Cells
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The SKOV3 and OVCAR-3 cells were seeded in 6-well plates at a density of 1×105 cells/well overnight. The SKOV3 and OVCAR-3 cells were then pre-incubated with or without 1 µM Fer-1 at 37°C for 1 h, followed by treatment with 10 or 20 µg/ml NCTD for a further 24 h at 37°C. Subsequently, 106 cells were treated in 100 µl RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) at 4°C for 15 min. The intracellular MDA and Fe2+ contents were then quantified using a Micro MDA assay kit (cat. no. BC0025; Beijing Solarbio Science & Technology Co., Ltd.) and Iron assay kit (cat. no. MAK025; Sigma-Aldrich; Merck KGaA), respectively, according to the manufacturer's instructions.
For the tumor tissues, MDA, ROS, GSH and Fe2+ levels were quantified using a Micro MDA assay kit (cat. no. BC0025; Beijing Solarbio Science & Technology Co., Ltd.), a Reactive Oxygen Species assay kit (cat. no. CA1401; Beijing Solarbio Science & Technology Co., Ltd.), a Micro Reduced Glutathione assay kit (cat. no. BC1175; Beijing Solarbio Science & Technology Co., Ltd.) and an Iron assay kit (cat. no. MAK025; Sigma-Aldrich; Merck KGaA), respectively, according to the manufacturer's instructions.
For the tumor tissues, MDA, ROS, GSH and Fe2+ levels were quantified using a Micro MDA assay kit (cat. no. BC0025; Beijing Solarbio Science & Technology Co., Ltd.), a Reactive Oxygen Species assay kit (cat. no. CA1401; Beijing Solarbio Science & Technology Co., Ltd.), a Micro Reduced Glutathione assay kit (cat. no. BC1175; Beijing Solarbio Science & Technology Co., Ltd.) and an Iron assay kit (cat. no. MAK025; Sigma-Aldrich; Merck KGaA), respectively, according to the manufacturer's instructions.
5
Soil Bacteria Response to Selenium
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Two typical soil microorganisms, namely, Gram-negative bacteria Bacillus sp. and Gram-positive bacteria E. coli, were chosen to investigate responses of different soil bacteria to exogenous Se. Briefly, the two bacteria were inoculated in a Luria-Bertani (LB) medium at a ratio of 1:100. SeNPs or SeO32– at a concentration of 0, 0.1, 0.5, 1, 5, and 10 mmol/L were added, respectively, and the mixed media were incubated at 37°C, 180 rpm for 24 h. OD600 was measured to characterize the biomass. Reactive oxygen species level was determined using Reactive Oxygen Species Assay Kit (Solarbio, Shanghai, China) following the manufacturer’s instructions. Glutathione peroxidase (GSH-Px) activity was determined using a Micro Glutathione Peroxidase Assay Kit according to the manufacturer’s protocol (Solarbio, Shanghai, China). Protein concentrations were determined following the bicinchoninic acid assay method (Smith et al., 1985 (link)).
6
ROS and MDA Levels Quantification
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Reactive oxygen species assay kit (CA1410; Solarbio) was used to assess the ROS level in cells. HEK293T cells and HeLa cells as well as stable BRD4 knockdown cells were treated with DMSO or BRD4 inhibitors for 24 h respectively. Rosup, a reagent that can stimulate cells to produce ROS, was diluted to 50 ug/mL by medium, and then added into the cells of Rosup group for 40 min. Then all cells of each group were washed twice with medium and the remaining liquid was sucked away. 100 μL ROS probe DCFH-D with a concentration of 5 μmol/L was added to cells of each group and incubated in a 37 ℃ cell incubator for 20 min. The cells were washed 3 times with serum-free culture solution. Fluorescence intensity was measured at excitation light 488 nm and emission light 525 nm. MDA levels were measured following the instructions of the MDA test kit (BC0025, Solarbio).
7
Reactive Oxygen Species Assay in KGN Cells
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The Reactive Oxygen Species Assay Kit (Solarbio, China) was used to determine ROS level. After COS treatment, KGN cells were loaded with 10 μM DCFH-DA in FBS-free medium for 20 min at 37°C. Then, the medium containing DCFH-DA was removed, and KGN cells were washed to remove unconjugated DCFH-DA.
8
Quantifying Intracellular Reactive Oxygen
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The generation of intracellular reactive oxygen species (ROS) was examined using a Reactive Oxygen Species Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, H9c2 cells (2×104 cells/well) were grown in 6-well plates and subjected to different treatments. The cells were collected and centrifuged at 2000 rpm/min for 5 minutes, and the supernatant was discarded. Then, the H9c2 cells were incubated with 5 μM 2,7-dichlorofluorescein diacetate (DCFH-DA) at 37°C for 20 minutes, washed three times with PBS, and resuspended in the flow tubes in 500 μL of PBS. After being resuspended, the cells were analysed by using a flow cytometer (BD FACSAria III, USA).
9
Cardiomyocyte Apoptosis and Oxidative Stress
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Rat cardiomyocytes (H9c2 cells) were purchased from the Cell Bank of the Chinese Academy of Sciences. The reagents and instruments used in this study are Trizol extraction solution (Thermo Fisher), reverse transcription kit (Thermo Fisher), Lipofectamine 3000 transfection reagent (Thermo Fisher), RIPA lysate, one-step TUNEL apoptosis assay kit (green fluorescence) (Shanghai Beyotime Biotechnology), DMEM medium (Hyclone, USA), fetal bovine serum (Gibco, USA), reactive oxygen species assay kit (Beijing Solarbio Life Sciences), PDE4D (Abcam, UK), cAMP (CST, USA), PKA (CST, USA), β-actin and HRP-labeled secondary antibody (Shanghai Beyotime Biotechnology), electrophoresis and transfer membrane reagents (Shanghai Beyotime Biotechnology), ECL luminescent solution (Millipore, USA), fluorescence microscope (Thermo Fisher), transfer electrophoresis tank (Bio-Rad, USA), and CO2 cell incubator (Thermo Fisher).
10
Triptolide Modulates Oxidative Stress and Apoptosis
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Triptolide was purchased from Chengdu Biopurify Phytochemicals Ltd. Cholesterol (Chol), distearoyl phosphatidylglycerole (DSPG) and egg yolk lecithin (PC-98T) were sourced from AVT (Shanghai) Pharmaceutical Tech Co., Ltd., Shanghai, China. GSH and GSSG assay kit, Annexin V-FITC/Propidium iodide (PI) Apoptosis Detection Kit, Cell Cycle and Apoptosis Analysis Kit, Bicinchoninic Acid (BCA) Protein Assay Kit and goat anti-rabbit IgG/HRP antibody were obtained from Beyotime Institute of Biotechnology, Nanjing, China. Reactive Oxygen Species Assay Kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. RPMI 1640 medium, trypsin, and fetal bovine serum (FBS) were provided by Gibco BRL, USA. Anti-caspase-3 antibody and anti-PARP-1 antibody were soured from Cell Signaling Technology, USA. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit monoclonal antibody was purchased from WuXi AppTec, Shanghai, China.
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