Seahorse analysis of isolated mitochondria

BK Beth Kelly
GC Gustavo E. Carrizo
JE Joy Edwards-Hicks
DS David E. Sanin
MS Michal A. Stanczak
CP Chantal Priesnitz
LF Lea J. Flachsmann
JC Jonathan D. Curtis
GM Gerhard Mittler
YM Yaarub Musa
TB Thomas Becker
JB Joerg M. Buescher
EP Erika L. Pearce
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Forty μg of mitochondria were loaded per well of a Seahorse XFp plate in 40 μl of mitochondrial assay buffer (MAS: 220 mM d-mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% w/v fatty acid-free BSA, pH 7.2)60 containing 10 mM malate, 10 mM glutamate, 4 mM ADP and 1,500 μM BHAM. The plate was centrifuged at 2,000g for 20 min at 4 °C. Then, 110 μl MAS containing malate, glutamate, ADP and BHAM was added to each well. Rotenone, succinate (10 mm), antimycin A or a combination of ascorbate (10 mM) and TMPD (100 μM) were injected as indicated.

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