This protocol outlines a high-throughput method to evaluate mitochondrial dysfunction in fibroblast mitochondria, indicating potential impacts on brain injury analysis.
This protocol describes the procedure for analyzing Electron Transport Chain activity of Complexes 1, 2, and 4 in mitochondria isolated from fibroblasts used by the Seifert Lab. It follows the "Electron Transport Chain Protocol" (ETCP) described in: Vekaria, H.J., Kalimon, O.J., Prajapati, P., Velmurugan, G.V., Sullivan, P.G., 2024. An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury. Front Mol Biosci 11, 1378536. which is a modified version of the "Respirometry in Frozen Samples" (RIFS) assay from: Acin-Perez, R., Benador, I.Y., Petcherski, A., Veliova, M., Benavides, G.A., Lagarrigue, S., Caudal, A., Vergnes, L., Murphy, A.N., Karamanlidis, G., Tian, R., Reue, K., Wanagat, J., Sacks, H., Amati, F., Darley-Usmar, V.M., Liesa, M., Divakaruni, A.S., Stiles, L., Shirihai, O.S., 2020. A novel approach to measure mitochondrial respiration in frozen biological samples. Embo j 39, e104073.
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Seifert et al. (2025) studied this question.
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