Mitochondrial ATP content was determined using the ATP Bioluminescence Assay Kit HS II (Roche Molecular Biochemicals, Indianapolis, IN). Freshly isolated mitochondrial pellets were extracted according to manufacturer’s instructions with some modifications (Singh, Englander, 2012 (link)), sonicated (Sonic Dismembrator Model 100, Fisher Scientific) and centrifuged at 16000 g for 8 minutes at 4°C. The supernatant was collected, diluted with Tris-acetate buffer pH 7.6 and 50 μl aliquots were transferred to black microtiter plates and mixed with equal volume of the luciferase reagent (ATP Bioluminescence Assay Kit HS lI). Measurements of luminescence were at 5-second integration time (TECAN Genios plate reader). ATP amounts were calculated from log-log graphs generated for ATP standard using Magellan software and normalized to mitochondrial protein.
Protocol detail
Mitochondrial ATP Content Quantification
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Acetate
Bioluminescence assay
Luciferase
Luminescence measurements
Mitochondrial
Mitochondrial protein
Pellets
Tris buffer
Corresponding Organization :
Other organizations : The University of Texas Medical Branch at Galveston
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Variable analysis
independent variables
- None explicitly mentioned
- Mitochondrial ATP content
- Freshly isolated mitochondrial pellets
- Tris-acetate buffer pH 7.6
- Centrifugation at 16000 g for 8 minutes at 4°C
- 5-second integration time for luminescence measurements
- Positive control: ATP standard used to generate log-log graphs
- Negative control: Not explicitly mentioned
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