NADPH oxidase activity in cell lysates was assayed using the lucigenin chemiluminescence assay according to methods described previously [18] (link) with modifications. The cells were treated with NSC23766 (50 μM) for 2 h and then exposed to high concentrations of glucose (30 mM) for the indicated period of time. After incubation, the cells were homogenized through sonication in PBS containing 1 mM MgCl2, 1 mM EGTA and protease inhibitors. The homogenates were centrifuged at 3000g for 10 min at 4 °C. The cleared lysates (250 μg/ml of protein) were then incubated with 20 μM lucigenin (Cayman Chemicals) and 100 μM NADPH (Sigma Aldrich) prepared in PBS. Chemiluminescence was measured every minute for 5 min using a luminometer. NADPH oxidase activity was expressed in relative light units (RLU) per μg protein. To detect the inhibitory effects of NADPH oxidase activity, cells were first incubated with VAS-2870 (10 μM) for 1 h. Subsequent steps followed the same procedures detailed above.
Lucigenin
Manufactured by Cayman Chemical
Sourced in United States
Lucigenin is a chemical compound used as a chemiluminescent detection agent in various laboratory applications. It emits light upon oxidation, which can be used to measure the presence or activity of specific molecules or enzymes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Fibrin coated 6 well plates, by Greiner (2 mentions)
Krebs buffer, by Cayman Chemical (2 mentions)
Nadph substrate, by Merck Group (2 mentions)
Nadph, by Merck Group (2 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Nadph, by Beyotime (1 mentions)
Troglitazone, by Cayman Chemical (1 mentions)
Ab124432, by Abcam (1 mentions)
Candesartan, by AstraZeneca (1 mentions)
Gw9662, by Cayman Chemical (1 mentions)
αsma fitc, by Merck Group (1 mentions)
Angiotensin 2, by Merck Group (1 mentions)
Gw0742, by Bio-Techne (1 mentions)
Apocynin, by Cayman Chemical (1 mentions)
6 protocols using lucigenin
1
Measuring NADPH Oxidase Activity in Cell Lysates
2
Assaying NADPH Oxidase Activity in BMDMs
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BMDMs were plated on 25 μg/ml fibrin-coated 6-well plates (Greiner Bio-One). 20 μg/ml 5B8 or IgG2b were added to the fibrin plates 2 h before plating cells. NADPH oxidase activity was assayed by the lucigenin-enhanced chemiluminescence method61 (link). Cells were collected by a cell scraper and homogenized in ice-cold Krebs buffer pH 7.4 (119 mM NaCl, 2.5 mM KCl, 1mM NaH2PO4, 1.3 mM MgCl2, 2.5 mM CaCl2, 11 mM glucose, and 20 mM HEPES, pH 7.4). The cell homogenate was centrifuged at 1000g, and the pellet was resuspended with luminescence buffer (Krebs buffer containing 10 μM lucigenin, Cayman Chemicals), before adding 100 μM NADPH substrate (Sigma-Aldrich). Luminescence was detected by an EnSpire microplate reader.
3
Assaying NADPH Oxidase Activity in BMDMs
4
Retinal NADPH Oxidase Activity Assay
5
Isolation and Measurement of NOX Activity in Adipocytes
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To determine the activity of NOX, the plasma membrane of mature 3T3-L1 adipocytes was isolated with the method reported by Chang Yeop Han and coworkers
[31] (link). Briefly, mature 3T3-L1 adipocytes were collected and incubated for 30 min with hypotonic lysis buffer (25 mM Tris-HCl, pH 7.4) containing 1 mM EDTA, 1 mM EGTA, and protease inhibitor cocktail (NCM Biotech, Suzhou, China), followed by homogenization with a Dounce homogenizer and centrifugation at 1000
g at 4°C for 10 min. Then, the supernatant was ultracentrifuged at 100,000
g for 1 h at 4°C. The pellet, which contained the plasma membrane, was dissolved in suspension buffer (50 mM triethanolamine, 150 mM NaCl, 2 mMMgCl
2, 0.1 mM EGTA, protease inhibitor cocktail). After protein concentration determination, equal amounts of protein were used to measure NOX activity. Then, lucigenin (5 μM; Cayman Chemical Company, Inc., Ann Arbor, USA) and NADPH (100 μM; Beyotime Biotechnology) were added and incubated for 10 min at 37°C. The chemiluminescence was recorded with a luminometer (Biotek, Winooski, USA).
[31] (link). Briefly, mature 3T3-L1 adipocytes were collected and incubated for 30 min with hypotonic lysis buffer (25 mM Tris-HCl, pH 7.4) containing 1 mM EDTA, 1 mM EGTA, and protease inhibitor cocktail (NCM Biotech, Suzhou, China), followed by homogenization with a Dounce homogenizer and centrifugation at 1000
g at 4°C for 10 min. Then, the supernatant was ultracentrifuged at 100,000
g for 1 h at 4°C. The pellet, which contained the plasma membrane, was dissolved in suspension buffer (50 mM triethanolamine, 150 mM NaCl, 2 mMMgCl
2, 0.1 mM EGTA, protease inhibitor cocktail). After protein concentration determination, equal amounts of protein were used to measure NOX activity. Then, lucigenin (5 μM; Cayman Chemical Company, Inc., Ann Arbor, USA) and NADPH (100 μM; Beyotime Biotechnology) were added and incubated for 10 min at 37°C. The chemiluminescence was recorded with a luminometer (Biotek, Winooski, USA).
6
Evaluating Vasoactive Pharmacological Agents
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The following drugs and doses were used: angiotensin II (Merck Millipore), 10 μg/kg for acute experiments, 1 mg/kg/day in minipumps, 10 -10 to 10 -5 M for myography; ATR1 antagonist (candesartan, Astra Zeneca), 10 mg/ml in drinking water; ACh (Sigma) 10 μg/kg in vivo, 10 -10 to 10 -5 M for myography, 5 µM for western blot analysis; SNP (Sigma) 200 μg/kg in vivo, 10 -10 to 10 -5 M for myography, 5 µM for western blot analysis; apocynin (Cayman Chemical) 1.5 mmol/L, added daily to the drinking water; troglitazone (Cayman Chemical) 10 μg/kg/day i.p. or 50 μg/kg/day oral gavage; GW9662 (Cayman Chemicals) 10 μg/kg/day i.p.; GW1229 (Adipogen) 10 μg/kg/day i.p. or 50 μg/kg/day oral gavage; GW0742 (Tocris) 5 μg/kg/day i.p. or 20 μg/kg/day oral gavage; lucigenin (Cayman Chemical) 5 µM. The following antibodies were purchased from Cell Signaling Technology: eNOS (rabbit polyclonal, 9572), phosphoeNOS (Ser1177, rabbit polyclonal, 9571), VASP (9A2, rabbit monoclonal antibody); other antibodies were phosphoVASP-biotinylated (Ser239, 16C2, mouse monoclonal, Enzo Life Sciences), RGS5 (chicken polyclonal, XW-7534, ProSci Inc.), αSMA-FITC (1A4, mouse monoclonal, Sigma), CD31 (rabbit polyclonal, ab124432, Abcam).
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