The cytotoxic effects of resveratrol (1.56-100 μM), doxorubicin (0.01-0.92µM) and their combination were analyzed in HUVECs by ATP assay kit (ATP Bioluminescent Somatic Cell Assay Kit; Sigma, Steinheim, Germany). Briefly, HUVECs were seeded at a density of 5×10 3 cells per well of 96-well plates in triplicate. The cells were treated with resveratrol (1.56-100 μM concentration), doxorubicin (0.01-0.92 µM) and their combinations for 48h. After 48h treatment, 150 μl of media was removed from each well and 50 μl of somatic cell ATP-releasing agent (ATP Bioluminescent Somatic Cell Assay Kit; Sigma, Steinheim, Germany) was added. The plate was well agitated and allowed to stand for 30 min at room temperature. At the end of the incubation, 50 μl of a mixture from each well was transferred to a white non-translucent plate. 50 μl of ATP Assay Mix (ATP Bioluminescent Somatic Cell Assay Kit, Sigma, Steinheim, Germany) was added to each well and the emitted light was measured using a 96-well Microplate Luminometer (Bio-Tek, Winooski, VT, USA). ATP viability assay was performed at least twice and the results are given as mean±SD of independent experiments.
Atp bioluminescent somatic cell assay kit
Manufactured by Merck Group
Sourced in United States, Germany
The ATP bioluminescent somatic cell assay kit is a laboratory equipment product that measures the levels of ATP (adenosine triphosphate) in somatic cells. It utilizes bioluminescence technology to quantify the ATP present in cell samples.
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57 protocols using atp bioluminescent somatic cell assay kit
1
Cytotoxic Effects of Resveratrol and Doxorubicin
2
Quantification of Intracellular ATP Content
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The intracellular ATP concentration was quantified using an ATP bioluminescent somatic cell assay kit (Sigma‒Aldrich). Briefly, fat bodies and flight muscles were obtained (10 days after the blood meal) and homogenized in PBS (100 µL), after which the intracellular ATP content was determined using an adenosine 5′- triphosphate (ATP) bioluminescent somatic cell assay kit (Sigma‒Aldrich) and an ATP standard curve, which was prepared for each experiment (Carvalho-Kelly et al., 2020 (link); Almeida-Oliveira et al., 2023 (link)). The final results were normalized to the amount of protein in the samples.
3
Hepatoprotective Mechanisms of Natural Compounds
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Aspirin, LPS, NAC, NADPH, reduced glutathione (GSH), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 5,5’-dithio bis-2-nitrobenzoic acid (DTNB), cytochrome c, coenzyme Q2, antimycin A, dodecyl maltoside, N-nitrosodimethylamine (NDMA), erythromycin and ATP bioluminescent somatic cell assay kits were purchased from Sigma (St Louis, MO, USA). 2,7-Dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Kits for mitochondrial membrane potential assays were procured from R & D Systems, MN, USA. Apoptosis detection kits for flow cytometry and IL6 and TNF-α measurement kits were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Polyclonal antibodies against beta-actin, HO-1, IκB-α, NF-κBp65, PARP, Nrf-2 and cytochrome c were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Reagents for electrophoresis and Western blot analyses were purchased from Bio-Rad Laboratories (Richmond, CA, USA).
4
Streptozotocin-Induced Diabetes Assay
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Streptozotocin (STZ), reduced and oxidized glutathione (GSH/GSSG), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NADH, NADPH, cytochrome c, coenzyme Q2, sodium succinate, antimycin A, dodecyl maltoside, resorufin, 7-ethoxyresorufin, methoxyresorufin, Hoechst 33342, and ATP bioluminescent somatic cell assay kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorofluorescein diacetate (DCFDA) was procured from molecular probes (Eugene, OR, USA). Kits for nitric oxide and caspase-3 and caspase-9 assays were purchased from R&D Systems Inc., MN, USA, and that for lipid peroxidation (LPO) from Oxis International Inc. (CA, USA). Kits for GSH/GSSG assay were procured from Promega Corp. (Madison, WI, USA). Apoptosis detection kits for flow cytometry were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). Rin-5F cells were obtained from American Type Culture Collection (Manassas, VA, USA). Polyclonal antibodies against beta-actin, caspase-3, PARP, NOS-2, Nrf2, GLUT 2, Bax, Bcl-2, Akt, and p-Akt were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Reagents for cell culture, SDS-PAGE, and Western blot analyses were purchased from Gibco BRL (Grand Island, NY, USA) and Bio-Rad Laboratories (Richmond, CA, USA).
5
Oxidative Stress and Inflammatory Response
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Aspirin, LPS, NAC, malondialdehyde, NADPH, 2-thiobarbituric acid, reduced glutathione (GSH), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 5,5′-dithio bis-2-nitrobenzoic acid (DTNB), cytochrome c, coenzyme Q2, antimycin A, dodecyl maltoside, N-nitrosodimethylamine (NDMA), erythromycin, 7-ethoxyresorufin, resorufin and ATP bioluminescent somatic cell assay kits were purchased from Sigma (St Louis, MO, USA). 2,7-Dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Kits for mitochondrial membrane potential assays were procured from R & D Systems, MN, USA. Apoptosis detection kits for flow cytometry and IL6 and TNF-α measurement kits were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). Murine macrophage J774.2 cells, which are circulatory monocyte macrophages from tumor bearing mice, were purchased from European Collection of cell cultures (Health Protection Agency Culture Collections, Salisbury, UK). Polyclonal antibodies against beta-actin, HO-1, TNF-α, Nrf-2, IκB-α, cytochrome c, and Bcl-2 and, NF-κBp65 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Reagents for electrophoresis and Western blot analyses were purchased from Bio-Rad Laboratories (Richmond, CA, USA).
6
Blastocyst ATP Quantification Protocol
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Individual blastocysts obtained from control, lipid-free and lipid-rich RN treatments were collected in 10 µL of PBS + 0.01% PVP and frozen at −80°C. ATP concentrations were determined by the ATP bioluminescent somatic cell assay kit (Sigma Chemical Co.) as previously described with minor modifications (Cukurcam et al. 2004 (link), Simsek-Duran et al. 2013 (link), Silva et al. 2015 (link)). Briefly, 1:5 diluted ATP assay mix was added to individual wells in an opaque 96-well plate. In a separate tube, somatic cell ATP-releasing reagent was mixed with each sample or standards, and added to the assay mix. The amount of light emitted was immediately measured using a Synergy 2 plate reader for luminescence (BioTek). ATP concentration was calculated by comparison to a standard curve ranging from 60 fmol to 2 pmol/100 µL and normalized (per cell) using the average total cell number for blastocysts from each treatment.
7
Metabolic Profiling of Cell Cultures
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Glucose consumption, lactate production, and PFK activity were measured using the Glucose Assay Kit, the Lactate Assay Kit, and the Phosphofructokinase Activity Colorimetric Assay Kit (all from Biovision, catalog nos. K686, K607, and K776, Milpitas, CA) according to the manufacturer's instructions, respectively. Intracellular adenosine triphosphate (ATP) levels and fructose‐6‐phosphate (F6P) levels were determined in cell lysates using the ATP Bioluminescent Somatic Cell Assay Kit and Fructose‐6‐Phosphate Assay Kit (Sigma–Aldrich, catalog nos. FLASC and MAK020) following the manufacturer's instructions.
8
Antibody and Reagent Acquisition for Cellular Analysis
9
Cellular ATP Content Quantification
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Relative cellular ATP content was measured using the ATP bioluminescent somatic cell assay kit (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Briefly, 2×105 cells were seeded in a 24-well plate. After 24h, cells were washed, centrifuged, and lysed. Lysates were collected, and luminescence was measured using a luminescence reader and normalized to the protein concentration. Measurements were performed in triplicates [9 (link)].
10
Quantification of Intracellular ATP in Epimastigotes
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The intracellular ATP was quantified by using an ATP bioluminescent somatic cell assay kit (Sigma-Aldrich). Briefly, mutant epimastigotes (107 parasites per tube, 0.1 ml) were incubated in a solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris–HCl (pH 7.2 adjusted with HCl). Cellular extracts were prepared by mixing 0.1 ml epimastigotes with 0.1 ml somatic cell ATP releasing reagent and the mixture was left on ice for 1 min. Half of the cellular extract (0.1 ml) was transferred to MTS-11C minitubes (Axygen, Union City, CA, USA) containing 0.1 ml ATP assay mix and stirred for 10 s at room temperature. The total amount of light emitted was measured with a GloMax Multi JR detection system (Promega, Madison, WI, USA). Total intracellular ATP concentration per cell number was calculated using a standard ATP curve, prepared, and analyzed in each experiment (Carvalho-Kelly et al., 2020 (link)).
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