Superoxide anion ( ) production was detected using a LumiMax Superoxide Anion Detection kit (Agilent Technologies, La Jolla, CA, USA) following the manufacture's protocol. Briefly, 50 μg sample proteins were suspended in 100 μl assay medium, and then mixed with 100 μl of reagent mixture containing 0.2 mM luminol and 0.25 mM enhancer. Light emissions were measured by a luminometer. content was expressed as relative light units (RLU)/μg protein/min.
Lumimax superoxide anion detection kit
Manufactured by Agilent Technologies
Sourced in United States
The LumiMax Superoxide Anion Detection kit is a product offered by Agilent Technologies. The kit provides a method for detecting and quantifying superoxide anion levels in biological samples.
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Lab products found in correlation
Lucetta luminometer, by Lonza (1 mentions)
May grunwald and giemsa, by Merck Group (1 mentions)
Brdu cell proliferation assay kit, by Cell Signaling Technology (1 mentions)
Caspase glo 3 7, by Promega (1 mentions)
Cycletest plus dna reagent kit, by BD (1 mentions)
Bca protein kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using lumimax superoxide anion detection kit
1
Superoxide Anion Production Assay
2
Luminol-Based Superoxide Quantification
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Superoxide anion production was measured using a standard luminol based LumiMax Superoxide anion detection kit (Agilent technologies, Santa Clara, CA)72 (link). The principle of the assay is that superoxide anion oxidizes luminol in a reaction that produces photons of light that are readily measured with a standard luminometer.
3
Measuring Superoxide in ER-HoxA9 Cells
4
Superoxide Anion Quantification in H9 Cells
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Generation of superoxide anion was determined using a Lumimax Superoxide Anion Detection Kit (Agilent Technologies, Santa Clara, CA, USA). H9 cells were trypsinized, washed with phosphate-buffered saline (PBS), and resuspended in fresh medium to incubate for 30 min at 37 °C. A total of 5 × 105 cells were incubated in superoxide anion assay medium including 0.1 mM luminol solution and 125 µM enhancer at room temperature for 30 min. The chemiluminescent light emissions of superoxide anion were measured with a Lucetta™ luminometer (Lonza, Basel, Switzerland).
5
Evaluating Dose-Dependent Cellular Responses
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A minimum of two biological replicates were evaluated for each assay. Dose response curves were generated using a 4-parameter equation (XLfit, IDBS). All kits were used according to the manufacturer’s recommendations. Cell cycle phase distribution was determined by flow cytometry using the Cycletest™ PLUS DNA Reagent Kit (BD Biosciences) and data were analyzed using FlowJo software (TreeStar Inc.). Proliferation was assessed using BrdU Cell Proliferation Assay Kit (Cell Signaling Technology) with a 4-hour pulse. Values were expressed as percent of vehicle. Caspase activity was measured using Caspase-Glo 3/7 (Promega). Luminescence values were normalized to CTG (Promega) for cell number. Peak activation was determined for MOLM-13, OCI-AML3, MV-4-11, THP-1, SIG-M5, HL-60, and Kasumi-1 to be days 4, 6, 6, 3, 4, 5, and 5, respectively. Values were expressed as a fold change relative to vehicle. Gene expression was evaluated using real time-quantitative polymerase chain reaction (RT-qPCR), as previously described.11 (link) Superoxide anion production was measured using LumiMax Superoxide Anion Detection Kit (Agilent Technologies). Values were expressed as fold increase over vehicle. Morphology was visually assessed after staining with May-Grunwald and Giemsa (Sigma).
6
Measuring Colonic Superoxide Anion
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Colonic tissues were collected immediately and homogenized in ice-cold PBS, and centrifugation at 12,000 × g at 4°C for 30 min and kept at −80°C until they were assayed. Superoxide anion (O2·−) production was detected using a LumiMax Superoxide Anion Detection kit (Agilent Technologies, La Jolla, CA, United States) according to the manufacturer’s protocol. Briefly, 50 μg colon proteins were suspended in 100 μl assay medium and then mixed with 100 μl detection reagent containing 0.2 mM luminol and 0.25 mM enhancer. The luminescence was measured using a luminometer, and the O2·− content was expressed as relative light units (RLU)/μg protein/min. The total protein content was determined by a BCA™ protein kit (Thermo, MA, United States).
7
Macrophage Superoxide Anion Assay
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NADPH oxidase activity was measured by chemiluminescence in macrophage cell cultures using a Lumimax Superoxide Anion Detection Kit (Agilent Technologies, Santa Clara, CA), as per the manufacturer's instructions.
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