ROS content was evaluated by utilising a DCFH-DA fluorescent probe (MAK143; Sigma-Aldrich). The H2O2 in cells can oxidise DCFH-DA to a highly fluorescent compound, dichlorofluorescein (DCF). After diverse treatments, hippocampal neurons were exposed to 10 mM DCFH-DA (Sigma-Aldrich) for 30 min. After washing, the fluorescence of the hippocampal neurons was assessed by a microplate reader (Thermo Fisher Scientific) or a fluorescence microscope (Leica).
Mak143
Manufactured by Merck Group
Sourced in United States
MAK143 is a laboratory instrument used for analysis and measurement purposes. It functions as a device to perform specific tasks within a research or testing environment. The core purpose of MAK143 is to provide accurate and reliable data, without interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Dcfh da, by Merck Group (1 mentions)
Fluorospectro photometer, by Thermo Fisher Scientific (1 mentions)
S0103, by Beyotime (1 mentions)
S0056, by Beyotime (1 mentions)
Glutathione (gsh), by Beyotime (1 mentions)
Fluorescence microplate reader, by Agilent Technologies (1 mentions)
Bb 470532, by BestBio (1 mentions)
Flow cytometry, by Agilent Technologies (1 mentions)
9 protocols using mak143
1
Quantifying Oxidative Stress in Hippocampal Neurons
2
Intracellular ROS Measurement in HASMCs
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ROS was measured by using a fluorometric intracellular ROS kit (MAK143, sigma). HASMCs were incubated with 10 µM DCFH-DA for 1 h. After washing with PBS three times, fluorescence signals were observed by using a fluorescence microscope.
3
Quantifying Intracellular ROS Levels
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The fluorimetric ROS assay MAK143 (Sigma Aldrich) was performed to examine the intracellular amount of ROS in the indicated cell lines. 1.5 × 104 cells/well were seeded out in black Cellstar® 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and treated with cisplatin, auranofin or MC3 in either subtoxic concentrations (1 µM of cisplatin, 0.1 µM of auranofin and 0.01 µM of MC3) or at IC50 of the corresponding cell line using DPBS as control. The assay was performed according to the manufacturer’s instructions. Fluorescence was analysed using a FLUOstar™ OPTIMA fluorescence scanner (BMG Labtech GmbH, Offenburg, Germany), with a 485 nm excitation and 520 nm emission after 4 h and 24 h of incubation. Finally, the ratio of total ROS measurement after 24 h and 4 h was calculated for each cell line and treatment.
4
Quantifying Intracellular ROS Levels
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The total ROS level was measured using a ROS assay kit (cat. no. MAK143; Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol. Briefly, intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 2V, 7V-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) using a fluorospectrophotometer (Thermo Fisher Scientific, Inc.) at an excitation wavelength of 488 nm and an emission wavelength of 535 nm.
5
Measuring Oxidative Stress in BMDMs
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For ROS detection by flow cytometry, BMDMs were pretreated with NAC (10 mM) for 4 h, then stimulated with LPS (100 ng/ml) and IFN-γ (20 ng/ml) for 4 h followed by incubation with FITC-ROS (MAK143, Sigma) at 37 °C for 45 min in the dark. BMDMs were treated with 1000 μM H2O2 for 10 min as a positive control.
For ROS detection by fluorescent staining, BMDMs were pretreated with NAC (10 mM) for 4 h, then stimulated with LPS (100 ng/ml) and IFN-γ (20 ng/ml) for 4 h followed by incubation with DHE (D1168, Invitrogen) for 30 min and Hoechst (C1027, Beyotime) for 10 min at 37 °C in the dark. BMDMs were treated with 500 μM H2O2 for 30 min as a positive control.
For ROS detection by fluorescent staining, BMDMs were pretreated with NAC (10 mM) for 4 h, then stimulated with LPS (100 ng/ml) and IFN-γ (20 ng/ml) for 4 h followed by incubation with DHE (D1168, Invitrogen) for 30 min and Hoechst (C1027, Beyotime) for 10 min at 37 °C in the dark. BMDMs were treated with 500 μM H2O2 for 30 min as a positive control.
6
Intestinal Oxidative Stress Biomarkers
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The supernatant was obtained after homogenization of intestinal tissue. SOD (Beyotime, S0103), MDA(Beyotime, S0131S) and GSH(Beyotime, S0056) levels were measured according to the reagent seller’s instructions(Beyotime Biotechnology, Shanghai, China.). The content of reactive oxygen species (ROS) in intestinal tissue was detected by DHE fluorescent probe (Sigma-Aldrich, MAK143). The fresh intestinal tissue was embedded and cut into 7 μm slices. The sections were incubated with DHE solution at 37°C for 30 min in dark. Put the slide into PBS(PH7.4) and shake it 3 times for 5 min each time. The tissue was then incubated at room temperature with DAPI solution for 10 min. Finally, the slides were examined under a fluorescence microscope.
7
Quantifying NK Cell ROS Production
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NK cells were sorted by FACS and seeded onto 24-well plates, followed by incubation with 1x ROS staining solution (MAK143, Sigma, MO, US) for 60 min at 37°C, 5% CO2. Subsequently, the NK cells were stimulated with 0.5 μg/mL LPS for 24 h. The intensity of ROS was analyzed by flow cytometry.
8
ROS Intracellular Fluorescent Assay
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The ROS Intracellular Fluorometric Kit especially detects superoxide and hydroxyl radicals in living cells. ROS react with the fluorogenic sensor (2,7 dichlorofluorescein diacetate (DCFH2-DA) resulting in a green fluorescent product. The assay was carried out following the manufacturer's recommendations (Sigma-Aldrich MAK 143). Twenty microliters of ROS detection reagent reconstituted and frozen at -20°C was added to 10 ml of the test buffer to form the fluorescence reaction solution (stable for 2 h). To the incubated plates, 50 μl of the fluorescence solution was added; they were kept 1 h at 37°C, and finally the fluorescence was detected using a fluorescence microscope.
9
Intracellular and Xenograft ROS Measurement
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2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining (MAK143, Sigma, USA) was used to measure intracellular ROS concentrations. A tissue ROS assay kit (BB-470532, BestBio, Shanghai, China) was used to measure xenograft tumor tissue ROS concentrations. In short, after the cells were treated with different concentrations of Rh4 for 48 hours or fresh xenograft tumor tissues were collected, the ROS concentration in cells and xenograft tumor tissues was measured by flow cytometry (Agilent, California, USA) or a fluorescence microplate reader (BioTek, Vermont, USA), respectively, according to the kit instructions. These experiments were performed in triplicate. The data are expressed as the percentage of the fluorescence intensity relative to that of the control group.
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