Ros glo kit

Manufactured by Promega

Sourced in United States

The ROS-Glo kit is a tool designed to measure the levels of reactive oxygen species (ROS) in biological samples. It provides a quantitative assay for detecting and monitoring ROS generation in cells or cell lysates. The kit utilizes a specific ROS-sensitive substrate that, upon oxidation, emits a luminescent signal proportional to the ROS concentration in the sample.

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Lab products found in correlation

Spectramax m5, by Molecular Devices (1 mentions) Gsh glo kit, by Promega (1 mentions) Mitosox red, by Thermo Fisher Scientific (1 mentions) Infinite m200 pro plate reader, by Tecan (1 mentions) Victor x5 luminometer, by PerkinElmer (1 mentions) Griess reaction kit, by Promega (1 mentions) Lopac1280 library, by Merck Group (1 mentions) Enspire, by PerkinElmer (1 mentions) Z vad fmk, by R&D Systems (1 mentions) Caspase glo 3 7 assay, by Promega (1 mentions)

7 protocols using ros glo kit

Quantifying Endogenous ROS Levels

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Endogenous ROS levels were measured using a ROS-GLO kit (Promega, Wisconsin, USA) according to manufacturer’s protocol. All four conditioned groups were plated at 2 x 104 cells per well in a 96 well plate and left in the incubator for 24 hours to adhere, prior to ROS detection. Luminescence was measured using a Spectramax M5 plate reader (Molecular Devices, California, USA).

Balouch B., Nagorsky H., Pham T., LaGraff J.T, & Chu-LaGraff Q. (2021). Human INCL fibroblasts display abnormal mitochondrial and lysosomal networks and heightened susceptibility to ROS-induced cell death. PLoS ONE, 16(2), e0239689.

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Mitochondrial Superoxide and ROS Quantification

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MitoSOX Red (catalog no. M36008, Thermo Fisher Scientific) was used to measure mitochondrial superoxide. Total ROS levels were measured using a ROS-Glo Kit (catalog no. G8820, Promega) and glutathione levels were measured using a GSH Glo kit (catalog no. V6911, Promega, Wisconsin, USA).

Whitburn J., Rao S.R., Morris E.V., Tabata S., Hirayama A., Soga T., Edwards J.R., Kaya Z., Palmer C., Hamdy F.C, & Edwards C.M. (2022). Metabolic profiling of prostate cancer in skeletal microenvironments identifies G6PD as a key mediator of growth and survival. Science Advances, 8(8), eabf9096.

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Measuring Cellular H2O2 Levels

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To measure overall H₂O₂ levels, a ROS-Glo kit (Promega, Madison, WI, USA) was used as described previously [31 (link)]. Cells were treated in PBS or MnTE-2-PyP (30 µM) for 24 h. Then, cells were re-seeded in Nunclon™ 96 Flat White Plates (Thermo Fisher Scientific, Rochester, NY, USA). H₂O₂ levels were measured according to the manufacturer’s instructions. The luminescence signal of H₂O₂ was recorded by an Infinite M200 Pro Plate Reader (Tecan, Männedorf, Switzerland).

Zhu Y., Kosmacek E.A., Chatterjee A, & Oberley-Deegan R.E. (2020). MnTE-2-PyP Suppresses Prostate Cancer Cell Growth via H2O2 Production. Antioxidants, 9(6), 490.

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Quantifying Oxidative Stress and Nitric Oxide

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ROS was measured in culture supernatants (80 μl) of infected and control cells using ROS-glo kit following the manufacturer's instruction (Promega, Madison, WI, USA). WCM260 treated with 200 μM of peroxide served as a positive control. All measurements were done in triplicate. Luminescence was measured in a Victor X5 luminometer (Perkin Elmer, Waltham, MA, USA). Results were expressed in relative light units (RLU). iNOS was determined in the cell culture supernatants (100 μL) in triplicate with the Griess reaction kit (Promega). Free-cell culture medium served as a negative control. Serial nitrite concentrations from 0 to 100 μM were used to generate the calibration plot. Absorbance was measured at 540 nm.

Chauhan R, & Michalak T.I. (2020). Kinetics of DNA damage repair response accompanying initial hepadnavirus-host genomic integration in woodchuck hepatitis virus infection of hepatocyte. Cancer genetics, 244.

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Development and Screening of QSOX1 Enzyme Inhibitors

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A HTS assay was developed using reduced denatured RNAse A substrate [38 (link)]. Hydrogen peroxide produced in the QSOX1 reaction was detected using ROS-Glo kit (Promega, Madison WI) as a primary assay and HyPerBlu (Lumigen, Southfield MI) as a secondary assay per manufacturers instructions. Assays were optimized and reaction kinetic parameters were determined. The assays were miniaturized to final volume of 2 uL. HTS was also performed using the ROS-Glo assay with a substrate concentration of 80 uM RNAse A (that is close to K_m value of 122 μM). rQSOX1 protein was utilized at 10 nM concentration, > 20-fold over the limit of assay detection yet still on the linear portion of the enzyme-dependent activity. rQSOX1 was screened against compounds from the LOPAC¹²⁸⁰ library (Sigma-Aldrich, St. Louis MO USA) at 12.5 μM compound concentration. Compounds that demonstrated > 50% inhibition were re-tested using single-concentration in triplicate wells, followed by concentration-dependent confirmation in the primary and secondary QSOX1 assays. A luminescent assay for glucose oxidase (GOx) was used as a counter-screen. The GOx assay utilized glucose as a substrate was detected using ROS-Glo. Active and selective compounds were purchased in dry powder form, dissolved in DMSO and reconfirmed in the assays before use in the confirmatory assays utilizing the GOx counter-screen.

Hanavan P.D., Borges C.R., Katchman B.A., Faigel D.O., Ho T.H., Ma C.T., Sergienko E.A., Meurice N., Petit J.L, & Lake D.F. (2015). Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines. Oncotarget, 6(21), 18418-18428.

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ROS Detection Assay Using ROS-Glo Kit

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For analysis of ROS levels, a ROS-Glo kit (Promega, Madison, Wisconsin, USA) was used according to the manufacturer's instructions. Briefly, 5 × 10⁴ cells resuspended in 80 µL per well in a 96 flat bottom plate were incubated overnight at 37 °C. After that 5 ng of phorbol myristate acetate and H₂O₂ substrate solution (20 µL) were added and incubated at 37 °C for 4 h. Then, 100 µL of detection solution containing Luciferin was added. The assay detects intra and extra-cellular ROS production. Luminescence was measured in a Multimode plate reader EnSpire (PerkinElmer, Waltham, Massachusetts, USA). All experiments were performed in duplicate, and the results were expressed in luminescence units.

Calapodopulos N.V., Sawan-Mendonça M.M., da Silva M.V., Oliveira C.J., Weffort V.R., Rodrigues D.B, & Rodrigues Jr V. (2021). Association of recurrent upper respiratory tract infections with low production of oxygen intermediates in children. Jornal de Pediatria, 98(4), 399-405.

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Caspase-Dependent ROS Detection Assay

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Caspase 3/7 activity was measured using the Caspase-Glo^® 3/7 Assay (Promega, Madison, WI, USA). Briefly, the assay is based on a caspase 3/7 substrate resulting in a caspase-specific luminescence signal equivalent to the caspase 3/7 activity. ROS Quantification was performed using the ROS Glo™ Kit (Promega, Madison, WI, USA). Here, the H₂O₂ substrate reacts with H₂O₂ generating a Luciferin precursor which is converted to light emitting Luciferin through a detection solution. The luminescence signal is proportional to the H₂O₂ concentration. Panc-1 cells were treated with 25–50 μM SW43-DOX for 6 h. Pan-caspase blocking (25 μM Z-VAD-FMK; R&D Systems, Minneapolis, MN, USA) or ROS elimination (200 μg/ml α-Tocopherol) was started 1 h prior to treatment. Caspase 3/7 substrate was added onto cells after treatment medium removal and luminescence was measured 30 min thereafter. H₂O₂ substrate was added together with SW43-DOX. Six hours later, the detection solution was added and luminescence was measured 20 min thereafter. Luminescence was quantified using the multi-mode microplate reader. Caspase 3/7 activity was normalized to parallel measured viability to account for occurred cell death by treatment and by untreated control group. Each sample was tested in at least triplicates.

Ludwig J.M., Gai Y., Sun L., Xiang G., Zeng D, & Kim H.S. (2016). SW43‐DOX ± loading onto drug‐eluting bead, a potential new targeted drug delivery platform for systemic and locoregional cancer treatment – An in vitro evaluation. Molecular Oncology, 10(7), 1133-1145.

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