OPCs were plated in a Seahorse XF24 (Agilent) Extracellular Flux Analyzer culture plate in the culture medium overnight. 1 h before the assay, cells were switched to the Mitostress assay medium (Seahorse Biosciences) supplemented with 10 mM glucose, 2 mM glutamine and 1 mM sodium pyruvate, following the manufacturer’s instructions. Oxygen consumption rate (OCR) during the Mitostress assay was measured with the Seahorse XF24 (Agilent) Extracellular Flux Analyzer (Seahorse Biosciences) following the manufacturer’s instructions. Following the Mitostress assay, 1 μM oligomycin, 2.0 μM FCCP (Isogenic 2 and FUSP525L) or 2.5 μΜ FCCP (Isogenic 1 and FUSR521H) and 0.5 μM antimycin A diluted in assay medium were the final concentration in the wells. Once the run was finished, all the medium was removed from the well and all the cells were collected and counted by ChemoMetec cell counter 900–002 NucleoCounter.
Mitostress assay medium
Manufactured by Agilent Technologies
Sourced in United States
Mitostress assay medium is a specialized cell culture medium designed for the assessment of mitochondrial function and stress in various cell types. It provides a controlled environment to measure parameters related to mitochondrial activity, such as oxygen consumption, ATP production, and metabolic responses.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using mitostress assay medium
1
Mitochondrial Respiration Profiling of OPCs
2
Mitochondrial Respiration Assay in Cells
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Cells were plated in a Seahorse XF24 (Agilent) Extracellular Flux Analyzer culture plate in culture medium overnight. Prior to the assay, cells were switched to the Mitostress assay medium (Seahorse Biosciences) supplemented with 25 mM galactose, 2 mM glutamine and 1 mM sodium pyruvate, following the manufacturer’s instructions. OCR during the Mitostress assay were measured with the Seahorse XF24 (Agilent) Extracellular Flux Analyzer (Seahorse Biosciences) following the manufacturer’s instructions. Following the Mitostress assay, 1 µM oligomycin, 0.5 µM FCCP (HeLa shCTR, shPERK, shPERK + PERKK618A, WT and DKO) or 2.5 µM FCCP (CTR and p. W681X) and 0.5 µM antimycin A diluted in assay medium were the final concentration in the wells. Once the run was finished, all the medium was removed from the well and all the cells were collected in 10 µl of Laemli buffer for BCA-based protein quantification.
3
Mitochondrial Respiration Profiling in Cell Lines
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Cells were plated at a density of 30,000 (shCntl, shATG5) and 50,000 (shBNIP3) cells per well of an XFp Extracellular Flux Analyzer culture plate in culture medium overnight. Before the assay, cells were switched to the Mitostress assay medium (Seahorse Biosciences, North Billerica, MA, USA) containing either glucose or galactose (25 mM) and supplemented with 2 mM glutamine and 1 mM sodium pyruvate (Sigma‐Aldrich), following the manufacturer instructions. Real‐time extracellular acidification (ECAR) and oxygen consumption (OCR) during the Mitostress assay were measured with the XFp Extracellular Flux Analyzer (Seahorse Biosciences) following the manufacturer instructions. Following the Mitostress assay, 8 µM oligomycin, 4.5 µM FCCP and 5 µM antimycin A diluted in assay medium were added whenever appropriate. Once the run was finished, all the medium was removed from the well and all the cells were collected in 10 µl of Laemli buffer for BCA‐based protein quantification (as explained before).
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