After measurement of ADP‐stimulated respiration, the incubation buffer containing mitochondria was taken from the respiration chamber and immediately supplemented with ATP assay mix (diluted 1:5; Sigma‐Aldrich, St. Louis, MO). Mitochondrial ATP production was determined immediately and compared with ATP standards using a 96‐well white plate and a Cary Eclipse spectrophotometer (Varian, Mulgrave, Victoria, Australia) at 560 nm emission wavelength.28
Atp assay mix
Manufactured by Merck Group
The ATP assay mix is a laboratory reagent used to measure the concentration of adenosine triphosphate (ATP) in a sample. It contains the necessary components to enable the quantification of ATP through a luminescence-based detection method. The core function of the ATP assay mix is to provide a standardized and reliable means of detecting and quantifying ATP levels in various biological and chemical samples.
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Lab products found in correlation
Spectramax m3, by Molecular Devices (2 mentions)
Pyruvate kinase from rabbit muscle, by Merck Group (2 mentions)
Myokinase, by Merck Group (2 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
D glucose, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Cary eclipse spectrophotometer, by Agilent Technologies (1 mentions)
Resveratrol, by Bio-Techne (1 mentions)
Curcumin, by Merck Group (1 mentions)
Victor multilabel plate reader, by PerkinElmer (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
6 protocols using atp assay mix
1
Mitochondrial ATP Production Measurement
2
Mitochondrial ATP Production Assay
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After the oxygen consumption measurement, the mitochondria from the respiration chamber were collected and immediately supplemented with the ATP Assay Mix (diluted 1:5) (Sigma, Aldrich). Mitochondrial ATP production was determined in each sample and compared with ATP standards using a 96-well white plate and a spectrofluorometer (SpectraMax® M3, Molecular Devices, EUA, San Jose, CA, USA) at 560 nm emission wavelength [41 (link)].
3
Fluorescence-Based Kinase Assay
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RES was purchased from Tocris Bioscience (Minneapolis, MN, USA). CUR, 3-isobutyl-1-methylxanthine (IBMX), DMSO, d -glucose, ATP assay mix, calmodulin from bovine heart, myokinase (adenylate kinase) from rabbit muscle, pyruvate kinase from rabbit muscle, cAMP, ATP, AMP, CTP, PEP, dithiothreitol (DTT), Protease Inhibitor Cocktail, and all other chemicals used in this study were obtained from Sigma.
4
Resveratrol and Curcumin Bioactivity Analysis
5
Extracellular ATP Quantification Protocol
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The extracellular levels of ATP were measured with a luciferin-luciferase assay kit, as previously described (Cunha et al., 2000 (link); Madeira et al., 2015b (link)). Briefly, cell culture supernatants were collected and stored at −80°C until used. Cells were collected and protein concentration was determined by the bicinchoninic acid (BCA) method (Pierce Biotechnology). Supernatants were quickly defrosted and incubated with ATP assay mix (Sigma-Aldrich) in an opaque 96-well plate. ATP levels were measured using a VICTOR multilabel plate reader (Perkin Elmer). The ATP concentration in each sample was determined by interpolation with a standard curve obtained from an ATP stock solution and was normalized to the amount of protein. Results are presented as percentage of control.
6
Mitochondrial ATP production measurement
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After the measurement of ADP-stimulated respiration, the incubation buffer containing mitochondria was taken from the respiration chamber and immediately supplemented with the ATP Assay Mix (diluted 1 : 5) (Sigma, Aldrich). Mitochondrial ATP production after each respiration measurement was determined immediately and compared with ATP standards using a 96-well white plate and a spectrofluorometer (SpectraMax® M3, Molecular Devices, EUA) at 560 nm emission wavelength [27 (link)].
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