In order to detect the O2− production which is dependent on Nox, we mixed 10 μL of the protein samples collected from the H9c2 cells with 80 μL of a solution made of Krebs-HEPES buffer which also contained diethyldithiocarbamate (5 μM, Sigma) and deferoxamine (25 μM, Sigma) and followed this by the addition of the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH; ENZO, Alexis Corporation) to a concentration of 1 mM and the substrate NADPH (Beyotime Biotechnology) to a concentration of 20 μM, with or without manganese-dependent superoxide dismutase (SOD, 8000 U/mL; Beyotime Biotechnology). An electron spin resonance (ESR) spectrometer (Bruker, Germany) was used to analyze O2− production in the protein samples. In the homogenates, the SOD-inhibitable fraction of the signal showed the total O2− production, and the results were indicated as fold changes compared with controls.
Nadph
Manufactured by Beyotime
Sourced in United States
NADPH is a coenzyme involved in various metabolic processes. It functions as a reducing agent, providing electrons for enzymatic reactions. NADPH plays a crucial role in biosynthesis, energy production, and cellular defense mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Deferoxamine, by Merck Group (1 mentions)
Diethyldithiocarbamate, by Merck Group (1 mentions)
Esr spectrometer, by Bruker (1 mentions)
Deferoxamine, by Selleck Chemicals (1 mentions)
Humidified incubator, by Thermo Fisher Scientific (1 mentions)
Gsk 872, by Selleck Chemicals (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Z vad fmk, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Erastin, by Selleck Chemicals (1 mentions)
Liproxstatin 1, by Selleck Chemicals (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Insulin, by Beyotime (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ab232857, by Abcam (1 mentions)
Vdac1, by Abcam (1 mentions)
8 protocols using nadph
1
Measuring Superoxide Production in H9c2 Cells
2
Ferroptosis and Necroptosis Modulation in LUAD
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Human LUAD cell lines A549 and H1299 as well as human embryonic kidney cells HEK293T were obtained from the American Type Culture Collection (ATCC). LUAD cells were cultured in RPMI 1640 (Gibco, Amercia) medium supplemented with 10% fetal bovine serum (FBS, Gibco, Amercia), and NADK- stable knockdown cell lines received additional puromycin to a final concentration of 2.5 µg/mL. HEK293T cells were maintained in DMEM (Gibco, Amercia) medium supplemented with 10% FBS. All cell lines were cultured in a humidified incubator (Thermo Fisher Scientific) at 37 °C with 5% CO2. The information of inhibitors or reagents used to treat the cells are as follows: ferroptosis inducer Erastin (Selleck, 0-9 µM), ferroptosis inducer RSL3 (Selleck, 0-0.5 µM), necroptosis inhibitor GSK'872 (Selleck, 10 µM), apoptosis inhibitor Z-VAD-FMK (Selleck, 5 µM), ferroptosis inhibitor Liproxstatin-1 (Selleck, 10 µM), ferroptosis inhibitor Deferoxamine (DFO, Selleck, 20 µM), and NADPH (Beyotime, 200 µM).
3
Insulin Disulfide Reduction Assay for Thioredoxin Activity
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Trx activity was evaluated with insulin disulfide reduction assay, as previously reported (24 (link),25 (link)). NP40 protein lysis with PMSF (Beyotime Institute of Biotechnology) was added to the cells to extract the total protein. Lysate was incubated with 2 µl DTT active buffer composed of 50 mM HEPES (pH 7.6), 1 mM EDTA, 1 mg/ml BSA and 2 mM DTT solution (Beyotime Institute of Biotechnology) at 37°C for 20 min. Then, the reaction buffer containing 200 µl 1 M HEPES (pH 7.6), 40 µl 0.2 M EDTA, 40 µl 40 mg/ml NADPH (Beyotime Institute of Biotechnology) and 500 µl 10 mg/ml insulin (Beyotime Institute of Biotechnology) was added. The reaction was started by the addition of 10 µl recombinational Trx reductase (Abcam) and incubated for 20 min at 37°C. The reaction was terminated by the addition 0.5 ml 6 mol/l guanidine-HCl and 1 mmol DTNB (Beyotime Institute of Biotechnology) and absorbance at 412 nm was measured using an automatic microplate reader (ELX-800; BioTek Instruments, Inc.).
4
Mitochondrial Stress and Oxidative Damage
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N-acetylcysteine (NAC), trypan blue and MitoTEMPO were obtained from Sigma‒Aldrich (USA). Diphenyleneiodonium chloride (DPI), VAS2870, Mdivi-1, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazon(FCCP), lucigenin, bardoxolone and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from MCE(USA), and NADPH was purchased from Beyotime Biotechnology (China). MitoTracker™ Deep Red, MitotrackerFM Green and MitoSOX™ were obtained from Invitrogen (UK). Specific antibodies were purchased from different companies as follows: GAPDH, LC3B,Nrf2 (CST, USA), NOX4, P62, TOMM20, VDAC1, Cleaved caspase-3 (Abcam, UK), BCL-2, and BAX (Affinity, China). The mitochondrial stress test complete assay kit (ab232857) and extracellular oxygen consumption assay kit (ab197243) were obtained from Abcam(UK).
5
Neuroprotective Strategies for HIBD
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For viral infection in vivo, 6 days prior to HIBD, a vector containing TIGAR short hairpin RNA (LV-sh_TIGAR; 1×109 transduction units/mL; Genechem, Shanghai, China) or GSDMD short hairpin RNA (LV-sh_GSDMD; 1 × 109 transduction U/mL; Genechem) was infused into the left lateral ventricle and striatum (2 μL/injection site) (Li et al., 2014) of the neonatal rats. Nicotinamide adenine dinucleotide phosphate (NADPH; 2.5 mg/kg; Beyotime, Nantong, China) was injected into the left lateral ventricle of the rat brain before HIBD modeling.
For viral infection in vitro, the HAPI rat microglial cells were treated with medium containing LV-sh_TIGAR (MOI = 100), LV-sh_GSDMD (MOI = 100), or LV-sh-scramble (normal control; MOI = 100; Genechem), and the medium was replaced with regular medium after 12 hours of viral treatment. The cells were cultured for 3 days and then subjected to OGD/reoxygenation (OGD/R). NADPH (10 μM; Beyotime) was added before OGD and was present in the regular medium used for reoxygenation at 24 hours.
For viral infection in vitro, the HAPI rat microglial cells were treated with medium containing LV-sh_TIGAR (MOI = 100), LV-sh_GSDMD (MOI = 100), or LV-sh-scramble (normal control; MOI = 100; Genechem), and the medium was replaced with regular medium after 12 hours of viral treatment. The cells were cultured for 3 days and then subjected to OGD/reoxygenation (OGD/R). NADPH (10 μM; Beyotime) was added before OGD and was present in the regular medium used for reoxygenation at 24 hours.
6
Cellular Glutathione Peroxidase Activity Assay
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With reference to the manufacturer’s instructions, Cellular Glutathione Peroxidase Assay Kit with a kit for glutathione peroxidase (GPx) activity in tissues or other samples, NADPH (Beyotime Biotechnology, Jiangsu, China, S0056) got employed for cell detection. The cells were collected through a cell scraper and washed once with PBS. The cell sample, lysed with cell lysate, was then centrifuged at 12,000 g for 10 min at 4°C. The supernatant for identifying the enzyme activity was taken out. In the next stage, on a 96-well plate, the detection buffer was added to test the sample, and the GPx detection working solution in sequence was evenly mixed and incubated at room temperature for 15 min. Next, after the adding and mixing of peroxide reagent solution, attached with the appropriate enzyme label, the solution was put immediately at 25°C and room temperature. The measurement of A340 with an instrument or a trace ultraviolet spectrophotometer was conducted for 5 consecutive minutes.
7
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).
8
Bilirubin Production Assay in Cells
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HO activity was measured as previously described (Vanella et al. 2010) with minor modification (Fig. 1A,C). Cell homogenates were added to a reaction mixture containing 30 μM hemin (Sigma-Aldrich), 2 mg/ml rat liver cytosol, 1 mM Mgcl 2 , 3 unit glucose-6-phosphatase dehydrogenase (Sigma-Aldrich), 1 mM glucose-6-phosphate (Roche, Basel, Switzerland), 2 mM NADPH (Beyotime), and 0.1 M potassium phosphate buffer (pH 7.4). The reaction was conducted at 37°C in the dark for 30 min and terminated by addition of 1 ml of chloroform. The extracted bilirubin was defined as the difference between 464 and 530 nm (ε = 40 mM -1 cm -1 ). The results were expressed as nmol of bilirubin/5 × 10 6 cells/h.
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