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Nadp nadph glo

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The NADP/NADPH-Glo assay is a bioluminescent-based kit that measures the levels of NADP (nicotinamide adenine dinucleotide phosphate) and NADPH (the reduced form of NADP) in biological samples. It provides a sensitive and quantitative method for detecting these important metabolic cofactors.

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Lab products found in correlation

Nad nadh glo, by Promega (8 mentions) Gsh gssg glo, by Promega (3 mentions) White 96 well plate, by Greiner (2 mentions) Celltiter glo, by Promega (2 mentions) Gsh glo assay kit, by Promega (2 mentions) Base solution, by Merck Group (2 mentions) Nad nadh assay kit, by Promega (2 mentions) Clariostar plus, by BMG Labtech (2 mentions) Ros glo, by Promega (1 mentions) Nad nadh glo assay kit, by Promega (1 mentions) Nad nadh glo detection reagent, by Promega (1 mentions) Clariostar plus plate reader, by BMG Labtech (1 mentions) Synergy h1 multi mode reader, by Synergy Software (1 mentions) White walled clear bottom 96 well plate, by Corning (1 mentions) Gsh gssg glo assay, by Promega (1 mentions) Z1 coulter particle counter, by Beckman Coulter (1 mentions) Lactate glo, by Promega (1 mentions) Glucose glo, by Promega (1 mentions) Glucose uptake glo, by Promega (1 mentions) Nad nadh, by Promega (1 mentions)

19 protocols using nadp nadph glo

1

Measurement of NADPH/NADP+ and NADH/NAD+ Ratios

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NADPH/NADP+ and NADH/NAD+ ratios were determined using the NADP/NADPH-Glo and NAD/NADH-Glo assay kits (Promega) according to the manufacturer's instructions. Scr and shNNT cells were (1 × 104 cells/100 μL) were seeded into each well of a 96-well plate. The cells were then placed in a reduced-serum environment (0.5% serum) for 16 h and experiments were conducted using serum and phenol red-free medium. Cells were then treated with angiotensin II (500 nM) for the times indicated. NADPH/NADP+ and NADH/NAD+ were determined in independent experiments. Cells were lysed in 0.2 m NaOH, 1% (w/v) dodecyltrimethylammonium bromide followed by treatment with 0.4 m HCl at 60 °C for 15 min. After neutralization with 0.5 m Tris base, samples were then mixed with an equal volume of NADP/NADPH-Glo or NAD/NADH-Glo detection reagent (Promega) prepared immediately before use. Luciferin luminescence was measured continuously for 2 h using the CLARIOstar Plus plate reader (BMG Labtech). The slope of the luciferin signal from the linear range of the assay was used to determine relative NADPH/NADP+ and NADH/NAD+ concentration compared with a Scr control.
Rao K.N., Shen X., Pardue S, & Krzywanski D.M. (2020). Nicotinamide nucleotide transhydrogenase (NNT) regulates mitochondrial ROS and endothelial dysfunction in response to angiotensin II. Redox Biology, 36, 101650.
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2

Measuring NADPH/NADP+ Ratio in Neutrophils

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The ratio of NADPH/NADP+ (or NADH/NAD+) was calculated after measuring the individual concentrations of NADPH and NADP+ (or NADH and NAD+) with NADP/NADPH-Glo (or NAD/NADH-Glo) assays (Promega). Neutrophils (2 × 104) were cultured in white 96-well plates (Greiner) in high glucose DMEM medium containing DMSO vehicle, 0.5 μM NA-11, 5 μM LDC7559, 100 μM Apocynin, or 200 μM 6-aminonicotinamide for 30 min, and then treated with 50 nM PMA. Cells were lysed in plates by addition of 1% dodecyltrimethylammonium bromide (DTAB). To measure NADPH, half of the samples was heated at 60°C for 30 min. The other half was treated with HCl before heating to 60°C, to measure NADP+. All samples were neutralized before adding luciferase detection reagent, then incubated for 1 h with gentle shaking. Luminescence was recorded on a plate reader (Envision).
Amara N., Cooper M.P., Voronkova M.A., Webb B.A., Lynch E.M., Kollman J.M., Ma T., Yu K., Lai Z., Sangaraju D., Kayagaki N., Newton K., Bogyo M., Staben S.T, & Dixit V.M. (2021). Selective activation of PFKL suppresses the phagocytic oxidative burst. Cell, 184(17), 4480-4494.e15.
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3

Cell Viability and Metabolic Assays

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CellTiter-Glo (Promega, Madison, WI, USA) was used for cell viability assays (24 h after treatment) and ATP assays (at indicated time points during or after β-lap treatment). The following assays were purchased from Promega: GSH/GSSG-Glo, NAD/NADH-Glo, NADP/NADPH-Glo, and ROS-Glo and were used as directed. Unless otherwise noted, all raw luminescent values for treatment conditions were normalized to the signal from untreated cells (T/C). Standard curves were generated to ensure linearity.
Moore Z., Chakrabarti G., Luo X., Ali A., Hu Z., Fattah F.J., Vemireddy R., DeBerardinis R.J., Brekken R.A, & Boothman D.A. (2015). NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective, PAR-independent metabolic catastrophe and cell death induced by β-lapachone. Cell Death & Disease, 6(1), e1599-.
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4

Quantifying Nrf2-mediated Redox Regulation

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Whole kidney tissue from Nrf2+/+ or Nrf2−/− mice dosed with DMSO or CDDO-Me (n=6 per group) was homogenised, and NADPH or glutathione levels were determined using NADP/NADPH-Glo or GSH-Glo assay kits (Promega), as per the manufacturer’s instructions, with the exception that NADPH and GSH were quantified after 5 and 2 min incubations, respectively, of the homogenates with the relevant detection reagents. NADPH was normalized to total protein content. Glutathione content was normalized to tissue weight.
Shelton L.M., Lister A., Walsh J., Jenkins R.E., Wong M.H., Rowe C., Ricci E., Ressel L., Fang Y., Demougin P., Vukojevic V., O’Neill P.M., Goldring C.E., Kitteringham N.R., Park B.K., Odermatt A, & Copple I.M. (2015). Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney. Kidney international, 88(6), 1261-1273.
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5

Measuring Cellular Redox Ratios

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Cells were seeded into the white-walled clear bottom 96-well plates (Corning). On day two, ratios of NADPH/NADP+ and GSH/GSSG were measured with NADP+/NADPH-Glo and GSH/GSSG-Glo Assays (Promega) according to the manufacturer’s instructions. Luminescence was measured using a Synergy H1 Multi-Mode reader.
Kiesel V.A., Sheeley M.P., Donkin S.S., Wendt M.K., Hursting S.D, & Teegarden D. (2022). Increased Ammonium Toxicity in Response to Exogenous Glutamine in Metastatic Breast Cancer Cells. Metabolites, 12(5), 469.
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6

Quantifying Cellular Redox Ratios

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NADP+/NADPH and NAD/NADH ratios were measured using the NADP+/NADPH-GLO and NAD+/NADH assay kit (Promega) respectively following the manufacturer’s instructions. Briefly, 5 x 106 H9C2 cells or 30 mg of heart tissue were lysed in 200 μl or 300 μl respectively of base solution (Sigma). Half of the lysate was treated with 0.4 N HCl at 60 °C in order to remove the NADH and NADPH to allow measurement of NAD+ and NADP+. Cell and tissue lysates were mixed with kit detection reagents and after 30 min, NADP+/NADPH ratios were measured using CLARIOstar® Plus (BMG, Labtech).
Fernandez-Caggiano M., Kamynina A., Francois A.A., Prysyazhna O., Eykyn T.R., Krasemann S., Crespo-Leiro M.G., Vieites M.G., Bianchi K., Morales V., Domenech N, & Eaton P. (2020). Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy. Nature metabolism, 2(11), 1223-1231.
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7

Quantifying NAD+, NADP, NADPH, and ATP in Xenografted Cells

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150,000 live human melanoma cells or 10,000 live human haematopoietic cells were isolated from xenografted mice by flow cytometry as described above then the cells were pelleted by centrifugation. The pellets were lysed in buffers from BioVision kits designed to quantitate NAD+ or ATP levels. A colorimetric assay using an NAD+/NADH cycling reaction was used to detect NAD+ levels (NAD+/NADH Quantitation Colorimetric Kit, BioVison)53 (link) according to the manufacturer's instructions. NADP and NADPH levels were measured using a luciferase-based assay using a similar cycling reaction (NADP/NADPH-Glo; Promega). ATP levels were quantitated using a luciferase-based assay in which luciferase activity depends on ATP from the lysed cells (Biovision)54 (link).
Eskiocak U., Ramesh V., Gill J.G., Zhao Z., Yuan S.W., Wang M., Vandergriff T., Shackleton M., Quintana E., Johnson T.M., DeBerardinis R.J, & Morrison S.J. (2016). Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma. Nature Communications, 7, 12336.
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8

Measuring NADPH/NADP+ Ratio in Neutrophils

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The ratio of NADPH/NADP+ (or NADH/NAD+) was calculated after measuring the individual concentrations of NADPH and NADP+ (or NADH and NAD+) with NADP/NADPH-Glo (or NAD/NADH-Glo) assays (Promega). Neutrophils (2 × 104) were cultured in white 96-well plates (Greiner) in high glucose DMEM medium containing DMSO vehicle, 0.5 μM NA-11, 5 μM LDC7559, 100 μM Apocynin, or 200 μM 6-aminonicotinamide for 30 min, and then treated with 50 nM PMA. Cells were lysed in plates by addition of 1% dodecyltrimethylammonium bromide (DTAB). To measure NADPH, half of the samples was heated at 60°C for 30 min. The other half was treated with HCl before heating to 60°C, to measure NADP+. All samples were neutralized before adding luciferase detection reagent, then incubated for 1 h with gentle shaking. Luminescence was recorded on a plate reader (Envision).
Amara N., Cooper M.P., Voronkova M.A., Webb B.A., Lynch E.M., Kollman J.M., Ma T., Yu K., Lai Z., Sangaraju D., Kayagaki N., Newton K., Bogyo M., Staben S.T, & Dixit V.M. (2021). Selective activation of PFKL suppresses the phagocytic oxidative burst. Cell, 184(17), 4480-4494.e15.
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9

Quantifying Nrf2-mediated Redox Regulation

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Whole kidney tissue from Nrf2+/+ or Nrf2−/− mice dosed with DMSO or CDDO-Me (n=6 per group) was homogenised, and NADPH or glutathione levels were determined using NADP/NADPH-Glo or GSH-Glo assay kits (Promega), as per the manufacturer’s instructions, with the exception that NADPH and GSH were quantified after 5 and 2 min incubations, respectively, of the homogenates with the relevant detection reagents. NADPH was normalized to total protein content. Glutathione content was normalized to tissue weight.
Shelton L.M., Lister A., Walsh J., Jenkins R.E., Wong M.H., Rowe C., Ricci E., Ressel L., Fang Y., Demougin P., Vukojevic V., O’Neill P.M., Goldring C.E., Kitteringham N.R., Park B.K., Odermatt A, & Copple I.M. (2015). Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney. Kidney international, 88(6), 1261-1273.
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10

Cellular NAD/NADH and GSH/GSSG Quantification

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Cells grown for 24hrs in serum-free medium were processed for the NAD/NADH-Glo™, NADP/NADPH-Glo™, or GSH/GSSG-Glo™ assays (Promega) according to the manufacturer’s instructions. At the time of the assay, the cell numbers of 3 replicates plated in parallel with experimental samples were counted using the Z1 Coulter Particle Counter (Beckman Coulter Life Sciences) and the average cell number was used to normalize luciferase readings.
White S.M., Avantaggiati M.L., Nemazanyy I., Di Poto C., Yang Y., Pende M., Gibney G.T., Ressom H.W., Field J., Atkins M.B, & Yi C. (2019). YAP/TAZ inhibition induces metabolic and signaling rewiring resulting in targetable vulnerabilities in NF2-deficient tumor cells. Developmental cell, 49(3), 425-443.e9.
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