Motor neurons were cultured on 8-well Seahorse XFp miniplates (103025-100, Agilent Technologies). Prior to the Cell Mito Stress Test assay (103010-100, Agilent Technologies), the neuronal medium was changed to Seahorse XF DMEM medium pH 7.4, including the Seahorse XF supplements (glucose, pyruvate and glutamine) (103680-100, Agilent Technologies). The Seahorse injection ports on the sensor cartridge were filled with 1 µM oligomycin (Port A), 2 µM carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) (Port B) and 0.5 µM Rotenone/Antimycin A (Port C). All mitochondrial inhibitors were purchased from Agilent Technologies. During the run, the oxygen consumption rates were measured using the Seahorse XF HS Mini Analyzer (Agilent Technologies) and subsequently normalized to total protein levels and quantified following the manufacturer’s instructions.
Cell mito stress test assay
Manufactured by Agilent Technologies
The Cell Mito Stress Test assay is a laboratory tool designed to assess the mitochondrial function of cells. It provides measurements of key parameters related to cellular respiration and energy production.
Automatically generated - may contain errors
2 protocols using cell mito stress test assay
1
Mitochondrial Function in Motor Neurons
2
Mitochondrial Respiration Profiling in HeLa Cells
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Mitochondrial respiration was measured with a Seahorse XFp instrument (Agilent Technologies) and the Cell Mito Stress Test assay (103010-100, Agilent Technologies). HeLa cells were cultured in Seahorse XFp miniplates (103025-100, Agilent Technologies) for one night. Before the respiration measurements, cells were rinsed in PBS and changed to Seahorse XF DMEM medium pH 7.4 enriched with glucose, pyruvate and glutamine as described in the Mito Stress Test protocol. After the medium change, cells were cultured for 1 h at 37 °C in a CO2-free incubator. The Seahorse injection ports on the sensor cartridge were filled with oligomycin (1 μM), FCCP (2 μM) and Antimycin A/Rotenone (0.5 μM). The sensor cartridges were placed on top of the Seahorse miniplates containing the cells and placed in the Seahorse XFp instrument for real-time analysis. Data were analysed with Wave software (Agilent Technologies).
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