U373MG SSEA1+ cells were transduced with NNT retroviruses or transfected with siNNT as described above. After 72 h, cells were dissociated and seeded at 4.5 × 105 cells per well in black wells of 96-well plates (Thermo Scientific). One micromole of MitoXpress Xtra HS (Luxcel Biosciences, Cork, Ireland) was added and then overlaid by mineral oil at 30 °C. Fluorescence was measured using a SpectraMax microplate reader (Molecular Devices) at 30 °C. ATP was measured using an ATP assay mix solution (Sigma) according to the manufacturer’s instructions. One mirogram of total protein was used for each reaction in white well and luminescence intensity was measured by a SpectraMax microplate reader (Molecular Devices).
Atp assay mix solution
Manufactured by Merck Group
The ATP assay mix solution is a laboratory reagent used to detect and quantify the presence of adenosine triphosphate (ATP) in biological samples. It provides a standardized and reliable method for measuring ATP levels, which is a fundamental indicator of cellular energy and metabolic activity.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate, by Thermo Fisher Scientific (1 mentions)
Spectramax microplate reader, by Molecular Devices (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
Victor x4, by PerkinElmer (1 mentions)
Somatic cell atp releasing agent, by Merck Group (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
Synergy h1 microplate reader, by Synergy Software (1 mentions)
3 protocols using atp assay mix solution
1
Measuring Mitochondrial Function and ATP
2
ATP Quantification in Cell Lines
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SKBR3 cells, fibroblasts, and iPS cells grown with 10 mM 2-deoxy-D-glucose (Sigma-Aldrich) for 24 h were washed with ice-cold PBS and incubated with 100 μl of Somatic Cell ATP Releasing Agent (Sigma-Aldrich) for 5 min. Next, 50 μl of the cell extract was added into wells of a light-protected 96-well plate that was preloaded with 100 μl of ATP Assay Mix Solution (Sigma-Aldrich) and incubated for 3 min at RT. Subsequently, the luminescence was measured using a luminescence plate reader (VictorX4, PerkinElmer).
3
Quantification of Nucleotide Levels
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For nucleotide quantification, bound ADP or ATP were released from protein samples by a 10-min boiling at 92 °C and centrifugation at 14,000 × g for 15 min at 4 °C. The resulting supernatant was divided equally and diluted 2.5 times in pyruvate kinase reaction buffer (125 mM Tris-HCl pH 7.4, 2.5 mM MgSO4 and 5 mM phosphoenolpyruvate). 30 U pyruvate kinase (Sigma-Aldrich) (which converts ADP to ATP) or a same volume of 50% glycerol as control were added to the samples, which were then incubated at room temperature for 50 min. 100 μl of the reaction mixture was added to an equal volume of ATP Assay Mix solution (Sigma-Aldrich) and photon emissions were read immediately using a Synergy H1 microplate reader.
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