Fetal bovine serum (FBS) and 0.25% Trypsin-EDTA were obtained from Gibco (Gibco, Grand Island, NY, USA). The whole protein extraction kit and BCA protein content assay kit were purchased from KeyGEN Biotechnology (KeyGEN, Nanjing, China). CCK-8 assay kit (APExBIO, Houston, TX, USA), mTOR pathway inhibitor PP242 (APExBIO, Houston, TX, USA), ROS assay kit (Elabscience Biotechnology, Wuhan, China), sodium butyrate (Sigma-Aldrich, St. Louis, MO, USA), and MMP assay kit was purchased from Beyotime (Shanghai, China). SDS-PAGE gel kit (Solarbio, Beijing, China), GAPDH antibody (Cambridge, UK), and P62 antibody were obtained from Abcam (Cambridge, UK). mTOR antibody, AKT1 antibody, and FOXO antibody were purchased from Proteintech (Proteintech, Wuhan, China).
Ros assay kit
Manufactured by Elabscience
Sourced in United States, China
The ROS assay kit is a laboratory tool designed to measure reactive oxygen species (ROS) levels in samples. It provides a quantitative assessment of ROS, which are chemically reactive molecules containing oxygen. The kit includes necessary reagents and protocols to perform the ROS detection assay.
Automatically generated - may contain errors
Lab products found in correlation
P62 antibody, by Abcam (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Cck 8 assay kit, by Apexbio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium butyrate, by Merck Group (1 mentions)
Whole protein extraction kit, by Keygen Biotech (1 mentions)
Mmp assay kit, by Beyotime (1 mentions)
Sds page gel kit, by Solarbio (1 mentions)
Inverted fluorescence microscopy, by Zeiss (1 mentions)
Multiskan ex spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Recombinant nfκb p65 protein, by Active Motif (1 mentions)
Malondialdehyde mda, by Abcam (1 mentions)
Quercetin qur, by Merck Group (1 mentions)
Carboxymethylcellulose cmc, by Merck Group (1 mentions)
Protein carbonyl content assay kit, by Abcam (1 mentions)
Superoxide dismutase sod activity colorimetric assay kit, by Abcam (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Zeiss (1 mentions)
10 protocols using ros assay kit
1
Evaluating Protein Expression and Signaling
2
Quantifying Cerebral Oxidative Stress
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Reactive oxygen species (ROS) levels were detected 24 h after MCAO using a ROS assay kit (Elabscience Biotechnology Inc. Houston, TX, USA). We followed the single suspension method according to the manual provided by the manufacturer. The right cerebral cortex tissue (10 mg) was dissolved, treated with a cooled reagent working solution, and incubated with an enzyme solution at 37°C for 30 min to digest the cortical tissue. The digestion was stopped with a reagent 3 working solution, centrifuged at 500 g for 10 min, and the supernatant was discarded. They were washed with reagent 3 working solution, centrifuged at 500 g for 10 min, and mixed with reagent 1 working solution. The mixture was incubated with 5 mM 2’, 7’-dichlorodihydrofluorescein diacetate at 37°C for 1 h to produce 2’, 7’-dichlorodihydrofluorescein (DCF). They were centrifuged at 1,000 g for 10 min and the cells were collected. The remaining cells were washed with a reagent 3 working solution and fluorescence values were observed at an excitation wavelength of 500 nm and an emission wavelength of 525 nm using a spectrophotometer. The result was expressed as the ratio of the other groups to the intensity of vehicle + sham group. The intensity of vehicle + sham group was set to 1.
3
ROS Generation Assay in Alveolar Macrophages
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A ROS assay kit (Elabscience, China) was used to detect ROS generation in AM. Concisely, cell suspensions collected from the different groups were incubated with 10 µM 2′,7-dichlorodihydrofluorescein diacetate (DCFH-DA) protected from light at 37 ℃ for 30 min followed by washing with PBS buffer to remove excess fluorescence probe. Hoechst 33,342 was performed to stain the active cell nucleus. Inverted fluorescence microscopy (Carl Zeiss) and flow cytometry were applied to test the fluorescence intensities in AM.
4
ROS Generation Measurement via DCFH-DA
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An ROS Assay kit (Elabscience, Inc.) was applied to investigate ROS generation in cells. In brief, cells were collected and suspended in 2′7′-dichlorofluoroscein diacetate (DCFH-DA; 10 mM) at 5×106 cells/ml at 37°C for 30 min. Then, the fluorescent activity was measured by a multiple plate reader with excitation at 502 nm and emission at 525 nm.
5
Quantifying ROS in E. coli O157:H7
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The level of ROS in Escherichia coli O157: H7 cells were determined by ROS Assay Kit (Elabscience Biotechnology Co., Ltd.). The dye is 2′,7′-dichlorofluorescein diacetate (DCFH-DA), which is a fluorescent probe that can pass freely through the cell membrane. DCFH-DA can be deacetylated into dichlorofluorescein (DCFH) in the cells. DCFH can be oxidized to 2 ′, 7 ' - dichlorofluorescein (DCF) to reflect the level of ROS in the cells. Briefly, Escherichia coli O157: H7 cells (1 × 108 CFU/mL) were treated with different concentrations of AMAs at 37 °C for 2h. The cells were washed three times by centrifugation at 5000g for 10 min at 4 °C with PBS (pH 7.4), and then resuspended. The Escherichia coli O157: H7 cells were incubated with DCFH-DA (the final concentration of 10 mM) at 37 °C for 30 min, washed with PBS for three times and resuspended. Fluorescence intensity was measured by microplate reader (excitation wavelength: 485 nm; emission wavelength: 525 nm). The relative ROS level was expressed as the ratio of fluorescence intensity between the treatment group and the control group (Liu et al., 2018 (link)).
6
Measuring Cerebral Cortex ROS Levels
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The ROS assay was performed using a ROS assay kit (Elabscience© Biotechnology Inc. Houston, Texas, USA) according to the manual provided by the manufacturer. We measured the amount of ROS by single cell suspension method. The isolated right cerebral cortex tissue was dissolved with a lysis buffer. The dissolved tissues were mixed with a reagent 3 working solution and then reacted with an enzyme solution to digest the tissue at 37 °C for 30 min. The digestion process was stopped by adding the reagent 3 working solution to the mixture. The mixture was filtered, centrifuged at 1000× g and the supernatant was discarded. The obtained cortical cells were mixed with the reagent 1 working solution. Cells were reacted with 5 mM 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C for 1 h. This process leads to the conversion of DCFH-DA into the fluorescent product DCF. After incubation, the cells were washed with reagent 3 working solution and centrifuged at 1000× g for 10 min. The precipitated cells were scanned with Multiskan EX spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation wavelength of 500 nm and an emission wavelength of 525 nm. The obtained values were expressed as tissue relative DCF pmol/mg.
7
Quercetin and Carboxymethyl Cellulose Assays
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Quercetin (QUR) (Cat# Q4951) and Carboxymethyl Cellulose (CMC) (Cat# C5678-500G) were purchased from Sigma Aldrich (St. Louis, MO, USA). Colorimetric and ELISA kits to measure levels of Troponin-I (Cat# E4737), creatinine kinase-MB (CKMB) (Cat# E4608), interleukin-6 (Cat# K4145), superoxide dismutase (SOD) (Cat# E458), tumor necrosis factor-α (TNF-α) (Cat# K1052), reduced glutathione (GSH) (Cat. NO. K454), and malondialdehyde (MDA) (Cat# K454) were purchased from BioVision, CA, USA. ROS assay kit (Cat# E-BC-K138-F) was purchased from Elabscience, CA, USA. A recombinant NF-κB p65 protein (Cat# 31102) and ELISA kit to measure the nuclear activity of NF-κB p65 (Cat# 40096) were purchased from Active Motif, Tokyo, Japan. An ELISA kit to measure serum and tissue levels of ANG II (Cat# CSB-E04494r) was purchased from CUSABIO, TX, USA. ANG II cocktail inhibitor (Cat# 9000681) was purchased from Cayman Chemicals, USA.
8
Oxidative Stress Markers in Cardiomyocytes
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The protein Carbonyl Content Assay Kit (ab126287, Abcam), PicoProbe™ Reduced Glutathione (GSH) Assay Kit (K740, BioVision, Inc., Exton, PA, USA), Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (K335, Biovision) were applied for the determination of oxidative stress-related indicators in cardiomyocyte lysates. MitoSOX™ Red (M36008, Invitrogen Inc., Carlsbad, CA, USA) staining was performed in live cells to analyze mtROS production. Intracellular ROS levels were assessed by a ROS Assay kit (Elabscience, Wuhan, Hubei, China).
9
ROS Assay in Mouse Skeletal Muscle
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Mouse leg skeletal muscle ROS assays were conducted using a ROS Assay Kit (Cat# E-BC-K138-F, Elabscience Biotechnology Inc., Houston, TX, USA) according to the manufacturer’s instructions. The tissues were digested into a single-cell suspension via collagenase digestion. Then, 106 cells were incubated with 10 μM DCFH-DA reagent for 30 min. Following centrifugation at 1000 rpm for 5 min, the cell pellets were washed twice and resuspended in Reagent 3 before being analyzed in a FACS Calibur flow cytometer.
10
Quantifying Intracellular ROS in NSCLC
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ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China). Briefly, NSCLC cells after preprocessing were incubated with 10 µM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) protected from light at 37°C for 30 min, and then followed by washing with PBS to remove excess fluorescence probe. The active cell nucleus was stained using Hoechst 33342. Fluorescence microscopy (Zeiss) was applied to test the fluorescence intensities in NSCLC cells.
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