Isolation and Mitochondrial Bioenergetics Analysis

Mitochondria were isolated from the freshly excised hearts of anesthetized mice. Harvested heart ventricles were homogenized in mannitol-sucrose-ethylene glycol tetraacetic acid (EGTA) buffer containing 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, and 1 mM EGTA (pH 7.4) using a glass/Teflon Potter Elvehjem homogenizer^{32 (link),35 (link),38}. Mitochondria were isolated from the homogenized heart lysates using differential centrifugation. Fresh mitochondria were subjected to a mitochondrial bioenergetics stress test, as outlined below. Mitochondria for western blot analyses were lysed in Cell Lytic M buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors (Roche).

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Abdullah C.S., Aishwarya R., Alam S., Morshed M., Remex N.S., Nitu S., Kolluru G.K., Traylor J., Miriyala S., Panchatcharam M., Hartman B., King J., Bhuiyan M.A., Chandran S., Woolard M.D., Yu X., Goeders N.E., Dominic P., Arnold C.L., Stokes K., Kevil C.G., Orr A.W, & Bhuiyan M.S. (2020). Methamphetamine induces cardiomyopathy by Sigmar1 inhibition-dependent impairment of mitochondrial dynamics and function. Communications Biology, 3, 682.

Publication 2020

Cell Lytic M buffer protease and phosphatase inhibitors

Bioenergetics Buffer Cell m Centrifugation Ethylene glycol tetraacetic acid Heart Heart ventricles Hepes Inhibitors Mannitol Mice Mitochondria Mitochondrial Phosphatase Protease Stress test Sucrose Teflon Western blot analyses

Corresponding Organization :

Other organizations : Louisiana State University Health Sciences Center Shreveport, Cincinnati Children's Hospital Medical Center

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Variable analysis

independent variables

Isolation of mitochondria from freshly excised hearts of anesthetized mice

dependent variables

Mitochondrial bioenergetics stress test
Mitochondrial protein expression (western blot analyses)

control variables

Mannitol-sucrose-ethylene glycol tetraacetic acid (EGTA) buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, and 1 mM EGTA, pH 7.4)
Glass/Teflon Potter Elvehjem homogenizer
Differential centrifugation for mitochondrial isolation
Cell Lytic M buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors (Roche) for mitochondrial lysis

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