The cellular production of ROS was measured using a luminescence-based ROS Glo Assay (Promega Corporation, Fitchburg, WI, USA). Therefore, exposure of HepG2 spheroids to test substances (SiO2, menadione) occurred simultaneously with incubation of a ROS Glo substrate. Menadione, which generates ROS by redox cycling,51 (link) was used as a positive control. After 24 hours, ROS Glo detection reagent was added for detection of luciferin. After incubation for 20 minutes luminescence was measured with a plate reader (SpectraMax; Molecular Devices LLC, Sunnyvale, CA, USA).
Ros glo assay
Manufactured by Promega
Sourced in United States, United Kingdom
The ROS-Glo assay is a quantitative, cell-based assay designed to measure reactive oxygen species (ROS) levels in living cells. The assay utilizes a luminogenic substrate that emits light in the presence of ROS, allowing for the assessment of oxidative stress in various cell types.
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Lab products found in correlation
Gsh glo glutathione assay, by Promega (2 mentions)
Primary antibodies against β actin, by Merck Group (1 mentions)
Gsh gssg glo assay, by Promega (1 mentions)
Total ros superoxide detection kit, by Enzo Life Sciences (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Hrp conjugated streptavidin, by Abcam (1 mentions)
Rabbit anti calnexin, by Merck Group (1 mentions)
Thioltracker, by Thermo Fisher Scientific (1 mentions)
Envision 2104, by PerkinElmer (1 mentions)
Gsh glo assay, by Promega (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
H1 hybrid microplate reader, by Agilent Technologies (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Synergy htx plate, by Agilent Technologies (1 mentions)
Infinity m200 pro, by Tecan (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Caspase glo 3 7, by Promega (1 mentions)
96 well solid white plate, by Corning (1 mentions)
Victor multilabel plate reader, by PerkinElmer (1 mentions)
Fluostar omega plate reader, by BMG Labtech (1 mentions)
19 protocols using ros glo assay
1
ROS Quantification in HepG2 Spheroids
2
Measuring Oxidative Stress in Cell Assay
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Cells were seeded in a white-walled, clear-bottom 96-well plate at a concentration of 10,000 cells/well, in 70 μL culture medium. After 24 h of incubation at previously described conditions, each well was treated with 10 μL of tamoxifen or T15 in a final concentration of 0.5, 2.5, and 25 μM. Culture medium and DMSO (<1 vol%) were used as negative controls, and 600 nM of bortezomib (bort.) was used as a positive control due to its well-known oxidative stress-inducing property [98 (link),99 (link),100 (link)]. Each data represents the average of 3 parallel measurements. After 24 h incubation with the compounds, components of the ROSGlo assay (Promega, Southhampton, UK) were prepared and added to the cells according to the manufacturer’s instructions. Luminescence was measured 24 h upon treatment with FluoroskanTM FL Microplate Fluorometer and Luminometer (Thermo Scientific, Waltham, MA USA).
3
ROS Quantification in Cell Cultures
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For measurement of ROS from cell culture supernatant, ROS-Glo Assay (Promega, Mannheim, Germany) was used. 4x105 cells/well were used in a 24-well format. 6h before ending the experiment, 30μL H2O2 substrate was added to the cell culture medium. ROS levels were normalized relative to the cell number determined by Sulfrhodamine assay (S1 File ).
4
Hydrogen Peroxide and Glutathione Assays
5
Nanoparticle-Induced DNA Damage and Oxidative Stress
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Nanoparticles can cause DNA damage to human cells [22 (link)]. DNA damage in cells was assessed using an assay for γH2AX histone phosphorylation (Apoptosis, DNA Damage and Cell Proliferation Kit®, BD Biosciences). Nanoparticles can cause oxidative damage to cells that induces apoptosis [23 (link)]. Oxidative responses in VEC were measured with a Molecular Probes CellROX Green® assay (Invitrogen) that detects an oxidation-sensitive dye bound to DNA in the cells by flow cytometry and also the ROS-Glo™ assay (Promega) for H2O2 measurements by high content analysis. Superoxide production was measured (Total ROS/Superoxide Detection kit®, Enzo) and the amount of oxidized glutathione was also measured (GSH/GSSG-Glo Assay™, Promega), as a measure of oxidative damage responses in the cells.
6
ROS Generation and Modulation in Human Monocytes
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ROS generation was detected using the ROS-Glo™ assay (Promega, Southampton, UK) according to the manufacturer's instructions (39 (link)). Briefly, cells were stimulated for 2 h with Pam3, MSU, or soluble uric acid in the presence of 25 μM ROS-Glo H2O2 substrate. Stimulations were completed in serum-free Optimem cell culture media (Gibco, ThermoFisher). After 2 h incubation at 37°C and 5% CO2, ROS levels were determined following a 20 min incubation with the ROS-Glo™ detection reagent. Luminescence was measured by spectrophotometry (Biotek Synergy HT).
N-acetyl-L-cysteine (NAC) was used in some experiments to inhibit ROS in primary human monocytes. For these experiments, cells were preincubated for 1 h with NAC and then stimulated for 6 h with Pam3 ± MSU crystals. NAC concentration was maintained during the 6 h stimulation. ROS levels were determined by ROS-Glo™ and IL-1β secretion by ELISA.
N-acetyl-L-cysteine (NAC) was used in some experiments to inhibit ROS in primary human monocytes. For these experiments, cells were preincubated for 1 h with NAC and then stimulated for 6 h with Pam3 ± MSU crystals. NAC concentration was maintained during the 6 h stimulation. ROS levels were determined by ROS-Glo™ and IL-1β secretion by ELISA.
7
Measurement of Intracellular ROS in Cells
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Intestinal epithelial cells SKCO15 were a kind gift from Dr. Charles Parkos, and were maintained in high glucose DMEM containing 10% fetal bovine serum, additionally supplemented with non-essential amino acids and L -glutamine. The mouse Rac-1 antibody was purchased from BD Transduction. The HydroCy3 was obtained from LI-Core (sold as ROSstar 550), and the Thiol Tracker from Thermo Scientific and were both used as directed in the manufacturers’ instructions and images acquired using a standard fluorescence microscope with camera. The biotinylated iodoacetamide was obtained from Anaspec. Anti-mouse secondary antibody conjugated to HRP was obtained from GE, while the streptavidin conjugated HRP was obtained from Abcam. Rabbit anti-calnexin was purchased from Sigma-Aldrich. ROS-GLO assay was purchased from Promega and performed in a 96-well format following the manufacturer’s instructions on overnight cultures of bacterial supernatants.
8
Glutathione and ROS Assessment in Lung Lavage
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Levels of glutathione (GSH) and oxidized glutathione (GSSG) were assess using a GSH-Glo™ Glutathione Assay (Promega, Madison, WI). The protocol (384-well format) was modified from the manufacturer’s protocol (96-well format, Promega V6911). In brief, 2.5 µl of unclarified lung lavage samples (in duplicate) were treated with 2.5 µl GSH-Glo reagent with or without TCEP (final concentration = 1 mM). Following a 30 min incubation at room temperature, luciferin detection reagent (5 µl) was added, and luminescence signals were recorded on a plate reader (PerkinElmer EnVision 2104, 700 nm, 0.5 s exposure) 15 min later. For reactive oxygen species (ROS), samples were tested using a ROS-Glo Assay (Promega). Lung lavage samples were prepared as described above for GSH-Glo assays. Following a 60 min incubation at room temperature, luciferin detection reagent (5 µl) was added, and luminescence signals were recorded on a plate reader (PerkinElmer EnVision 2104, 700 nm, 0.5 s exposure) 20 min later.
9
Quantification of Cellular ROS and GSH
10
Oxidative Stress Assessment of RA-loaded Microspheres
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The ROS-Glo assay was performed to estimate the oxidative stress caused by the RA-loaded magnetite microspheres on the BHK—baby hamster kidney cell line. The samples were tested at the concentration of 0.05 mg/µL, at two time-points, i.e., 24 and 72 h. The H2O2 levels were measured using the ROS-Glo assay (Promega), according to the instructions of the manufacturer. Bioluminescent measurements were performed using a SpectraMax i3x Multi-Mode microplate reader and the SoftMax Pro 6 software and the results were expressed as record relative luminescence units (RLU).
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