Agilent
Seahorse Cell Mito Stress Test kit
103015-100
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We use the Agilent Seahorse Cell Mito Stress Test kit to assess mitochondrial oxygen consumption rate (OCR) in primary and immunomagnetically isolated astrocytes. We determine OCR at baseline and each point after adding oligomycin (inhibits ATP synthase [complex V]), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP; uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential), and rotenone (inhibits complex I).
Metabolic Flux Assay
Immunomagnetically isolated astrocytes
Optimization is required for the FCCP concentration.
The OCR assay uses probes loaded on a sensor cartridge for fluorometric detection of extracellular medium O2 flux changes of cells. We utilized Seahorse Cell Mito Stress Test kit to assess mitochondrial respiration in mu opioid receptor deficient astrocytes (Oprm1 icKO astrocytes) isolated from mice exposed to morphine. Before the assay, astrocytes were plated at a density of 50,000 per well in XF96 cell-culture microplates pre-coated with poly-d-lysine and incubated in AstroMACS cell medium for 4 days. On day 5, the medium was replaced with Seahorse XF medium supplemented with 10 mM D-glucose, 1 mM glutamine, and 1 mM sodium pyruvate, and OCR was measured using XF96 Extracellular Flux Analyzer (Agilent). Non-mitochondrial OCR was determined by OCR after injection of rotenone. Basal respiration was determined by starting level of cellular OCR subtracting the non-mitochondrial OCR. Maximal respiration was determined by maximum OCR rate after FCCP injection minus non-mitochondrial OCR. ATP production was determined by the last OCR measurement before oligomycin injection minus the minimum OCR measurement after oligomycin injection.
37408246
Consistent results are achieved through the use of high-quality reagents.
The kit comes with a high price.
The kit offers compounds of high quality, allowing for a comprehensive exploration of mitochondrial function.