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Nadph substrate

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NADPH substrate is a key component in biochemical and enzymatic reactions. It functions as a coenzyme, providing reducing power for various metabolic processes. The NADPH substrate serves as an essential cofactor for enzymes involved in biosynthesis, antioxidant defense, and energy production.

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Lab products found in correlation

Lucigenin, by Merck Group (2 mentions) Lucigenin, by Cayman Chemical (2 mentions) Fibrin coated 6 well plates, by Greiner (2 mentions) Krebs buffer, by Cayman Chemical (2 mentions)

4 protocols using nadph substrate

1

NADPH Oxidase Activity Luminescence Assay

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The enzymatic activity of NADPH oxidase was assessed by a luminescence assay in the presence of lucigenin (250 μm) and NADPH substrate (100 μm; Sigma, St Louis, MO, USA) as described (Liu et al., 2010). Fluorescence intensity was continuously monitored for 15 min with a TD20/20 luminometer. The chemiluminescent signal was corrected by the protein concentration of each sample homogenate.
Guo Y., Li P., Gao L., Zhang J., Yang Z., Bledsoe G., Chang E., Chao L, & Chao J. (2017). Kallistatin reduces vascular senescence and aging by regulating microRNA‐34a‐SIRT1 pathway. Aging Cell, 16(4), 837-846.
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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.
Ryu J.K., Rafalski V.A., Meyer-Franke A., Adams R.A., Poda S.B., Coronado P.E., Pedersen L.Ø., Menon V., Baeten K.M., Sikorski S.L., Bedard C., Hanspers K., Bardehle S., Mendiola A.S., Davalos D., Machado M.R., Chan J.P., Plastira I., Petersen M.A., Pfaff S.J., Ang K.K., Hallenbeck K.K., Syme C., Hakozaki H., Ellisman M.H., Swanson R.A., Zamvil S.S., Arkin M.R., Zorn S.H., Pico A.R., Mucke L., Freedman S.B., Stavenhagen J.B., Nelson R.B, & Akassoglou K. (2018). Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nature immunology, 19(11), 1212-1223.
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3

Assaying NADPH Oxidase Activity in BMDMs

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
Ryu J.K., Rafalski V.A., Meyer-Franke A., Adams R.A., Poda S.B., Coronado P.E., Pedersen L.Ø., Menon V., Baeten K.M., Sikorski S.L., Bedard C., Hanspers K., Bardehle S., Mendiola A.S., Davalos D., Machado M.R., Chan J.P., Plastira I., Petersen M.A., Pfaff S.J., Ang K.K., Hallenbeck K.K., Syme C., Hakozaki H., Ellisman M.H., Swanson R.A., Zamvil S.S., Arkin M.R., Zorn S.H., Pico A.R., Mucke L., Freedman S.B., Stavenhagen J.B., Nelson R.B, & Akassoglou K. (2018). Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nature immunology, 19(11), 1212-1223.
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4

Enzymatic NADPH Oxidase Activity Assay

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The enzymatic activity of NADPH oxidase was assessed by a luminescence assay in the presence of lucigenin (250 μm) and NADPH substrate (100 μm; Sigma, St Louis, MO, USA). Fluorescence intensity was continuously monitored for 15 min with a TD20/20 luminometer. The chemiluminescent signal was corrected by protein concentration of each sample homogenate.
Lee G., Hoang T., Jung E., Jung S., Han S., Chung M., Chae S, & Chae H. (2020). Anthocyanins attenuate endothelial dysfunction through regulation of uncoupling of nitric oxide synthase in aged rats. Aging Cell, 19(12), e13279.
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