Quantitative measurements of ROS was acquired with Muse® Oxidative Stress Kit (Merck Millipore, Germany). HaCaT cells were seeded at a density of 4 x 105 cells/well into 6-well plates in defined Keratinocyte-SFM medium (Gibco, Thermo Fisher Scientific, CA, USA) supplemented with a pituitary extract including BPE and EGF (Gibco, Thermo Fisher Scientific, CA, USA). After a 24-hour incubation, cells were cultivated in serum-free keratinocyte medium without growth supplements for 16 hours. For experimental analysis cells were pretreated with 100 μM genistein or 10 mM NAC for 2 hours, and then incubated with a combination of proinflammatory “cytokine mix” or TNF-α 10 ng/mL or LPS 1μg/mL (Gibco, Thermo Fisher Scientific, CA, USA; Sigma-Aldrich, St. Louis, USA) for 30 minutes. The control cells were left untreated. Trypsinized cells were collected, and the count and percentage of cells undergoing oxidative stress based on the intracellular detection of superoxide radicals were determined by MUSE® Cell Analyzer (Merck, Millipore, Germany) using a commercial kit Muse® Oxidative Stress Kit (Merck Millipore, Germany) according to the manufacturer’s instructions. An average of 10,000 cells were analyzed for each condition. Triplicate independent experiments were conducted.
Muse oxidative stress kit
Manufactured by Merck Group
Sourced in United States, Germany, France
The Muse Oxidative Stress Kit is a lab equipment product designed to measure oxidative stress in biological samples. It provides quantitative analysis of oxidized proteins, a key indicator of oxidative stress levels.
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Muse benchtop flow cytometer, by Merck Group (2 mentions)
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Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
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Menadione, by Merck Group (1 mentions)
Total reactive oxygen species assay kit, by Thermo Fisher Scientific (1 mentions)
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92 protocols using muse oxidative stress kit
1
Quantifying Oxidative Stress in HaCaT Cells
2
Oxidative Stress Evaluation of Cell Lines
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The SKOV-3 cells (1 × 105 cells/well, 12-well plates) were treated with the extract in the concentration range of 40.0–300.0 µg/mL. After 24 h of incubation, the cells were stained with a Muse Oxidative Stress Kit (Merck Millipore, Burlington, MA, USA) and analyzed with a Muse Cell Analyzer (Merck Millipore, Burlington, MA, USA). The experiments were done in three independent repeats.
The HaCaT cells (1 × 105 cells/well, 12-well plates) were treated with menadione (Merck Millipore, Burlington, MA, USA) dissolved in DMSO at concentrations of 100 and 200 μM, and with the extract over a concentration range of 40–200 µg/mL. The concentration of DMSO (control) in wells did not exceed 0.2 % (v/v). After 24 h, the cells were harvested and stained with reagents from a Muse Oxidative Stress Kit (Merck Millipore, Burlington, MA, USA) and analyzed with a Muse Cell Analyzer (Merck Millipore, Burlington, MA, USA). The experiments were done in three independent repeats.
The HaCaT cells (1 × 105 cells/well, 12-well plates) were treated with menadione (Merck Millipore, Burlington, MA, USA) dissolved in DMSO at concentrations of 100 and 200 μM, and with the extract over a concentration range of 40–200 µg/mL. The concentration of DMSO (control) in wells did not exceed 0.2 % (v/v). After 24 h, the cells were harvested and stained with reagents from a Muse Oxidative Stress Kit (Merck Millipore, Burlington, MA, USA) and analyzed with a Muse Cell Analyzer (Merck Millipore, Burlington, MA, USA). The experiments were done in three independent repeats.
3
Oxidative Stress Assay by Flow Cytometry
4
Measuring Oxidative Stress in MCF-7 Cells
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MCF-7 cells (2x106 cells/well) grown in 12-well plates were incubated with 125 µg/ml of NF or IR-NF for 24 h at 37˚C. The oxidative stress assay was conducted by quantitative measurement of cellular populations undergoing oxidative stress using the Muse Cell Analyser and Muse Oxidative Stress kit (MilliporeSigma). According to the manufacturer's protocol, the cells were detached, resuspended to obtain 1x106 cells/ml and incubated at 37˚C for 30 min with the Muse Oxidative Stress working solution. The number of oxidised cells were counted using the Muse Cell Analyser based on the intensity of the red fluorescence. The results were obtained from four independent experiments.
5
Quantifying Oxidative Stress in LCLs
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To determine superoxide production, untreated LCLs were stained with reagents contained in the Muse Oxidative Stress kit (Cat # MCH100111, Millipore) and fluorescence was detected using a Muse Cell Analyzer (Millipore) as per manufacturer’s protocol. Hydrogen peroxide production was determined in untreated LCLs utilizing a Total Reactive Oxygen Species (ROS) assay kit (Cat # 88-5930-74) from eBioscience as per manufacturer’s instructions.
6
Superoxide-Sensitive Fluorescent Probe Assay
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Dihydroethidium (DHE) has been shown to be oxidized by superoxide to form 2-hydroxyethidium (2-OH-E+) (ex 500–530 nm/em 590–620 nm) or by non-specific oxidation by other sources of reactive oxygen species (ROS) to form ethidium (E+) (ex 480 nm/em 576 nm). NRK52E cells (2 × 104) were incubated in 24-well plates and then treated with nifedipine (30 mM) for 16 h (peak ROS level) or with H2O2 (500 mM) for 30 min as the positive control. The cells were then dyed with the Muse Oxidative Stress Kit (MCH100111; Millipore, Billerica, MA, USA) and subjected to the Muse® Cell Analyzer (Luminex Corp., Austin, TX, USA). All measurements were repeated at least three times. The data were analyzed by the Muse Software module version 1.5.0.0.
7
Cytotoxicity and Apoptosis Assays
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MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), pifithrin-α (p53 inhibitor) and LY294002 (PI3K/Akt inhibitor), alliin, resveratrol and gallic acid were purchased from Sigma Aldrich (Sigma Aldrich, USA). LDH (The Pierce Lactate Dehydrogenase) Cytotoxicity Assay Kit was purchased from Thermo Fisher Scientific (Waltham, USA). Specific antibodies such as p-Akt (Ser473), (total form) Akt, p53, p-MDM2, Bcl-2, Bax, Bak, PARP, and β-actin were obtained from Cell Signaling Technology (Beverly, USA) and caspase-3(inactivation form and activation form) was purchased from Abcam (Cambridge, USA). Muse™ Annexin V and Dead Cell Assay kit, Muse™ MitoPotential Kit, Muse™ Cell Cycle Kit, Muse™ Oxidative Stress Kit, and Muse™ Cell Analyzer were purchased from Millipore (EMD Millipore Corporation, Germany). Apo-ONE Homogeneous Caspase 3/7 Assay Kit was purchased from Promega (Wisconsin, USA).
8
Oxidative Stress Assay for VPA Effect
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To determine the effect of VPA treatment on oxidative stress, the assay was performed using the Muse® Oxidative Stress Kit (Millipore, Burlington, MA, USA). SH-SY5Y cells were plated at 1 × 106 cells/mL in a 6-well plate and incubated in a 5% CO2 incubator at 37 °C for 24 h. The cells were treated with VPA (0, 1, 5, and 10 mM) for 24 h and then harvested by trypsinization, centrifuged, washed with phosphate-buffered saline, pelleted, and resuspended in 1 × assay buffer at 1 × 106 to 1 × 107 cells/mL. The cell suspension (10 μL) was mixed with 190 μL of working solution and incubated at 37 °C for 30 min. The working solution was prepared by diluting the kit’s reagent in 1× assay buffer, as described by the manufacturer. The mixtures were vortexed for 3–5 s and analyzed using a MuseTM cell analyzer (Millipore, Burlington, MA, USA).
9
Quantification of Cardiomyocyte Oxidative Stress
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The detection of cardiomyocytes undergoing oxidative stress was performed using Muse Oxidative Stress Kit (Millipore, USA). According to the manufacturer’s protocol, treated cells were incubated with dihydroethidium (DHE), which is a cell mem-brane-permeable redox-sensitive probe that reacted with superoxide anions and formed DNA-binding fluorophore. The percentage of cells with reactive oxygen species (ROS + ) or without reactive oxygen species (ROS-) were quantified using Muse Cell Analyzer (Millipore, USA).
10
Quantifying Oxidative Stress in Cells
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Percentages of cells exhibiting oxidative stress were determined using a Muse Oxidative Stress Kit (#6C3598, Millipore, Billerica, MA, USA). Briefly, 10 μl of cell suspension in 1X Muse assay buffer was added to 190 μl of Muse Oxidative Stress working solution and incubated in the dark for 30 min at room temperature. The analysis was then performed using a Muse Cell Analyzer (Millipore, Billerica, MA, USA).
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