Basal respiration rates of mitochondria were measured via previously reported methods using a kit (MitoXpress® Xtra Oxygen Consumption Assay; Agilent Technologies, Inc.) (31 (link)). In brief, C2C12 myoblasts previously seeded into 96-well microplates (Corning, Inc.) were incubated for 5 day to allow them to differentiate. C2C12 myotubes were then treated with various concentrations of DEX (0, 25, 50, 100 or 200 µM) for 24 h, 50 µM DEX for 0, 3, 6, 12 or 24 h or S-Rg3 (0, 0.02, 0.2 or 2 µM) and/or DEX (50 µM) for 6 h, all at 37°C. OCRs were measured using a plate reader at room temperature (Cytation 5; BioTek Instruments, Inc.).
Mitoxpress xtra oxygen consumption assay
Manufactured by Agilent Technologies
The MitoXpress Xtra Oxygen Consumption Assay is a fluorescence-based tool for measuring oxygen consumption in cell cultures and tissue samples. It provides a real-time, non-invasive method for quantifying oxygen levels in cell-based assays.
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3 protocols using mitoxpress xtra oxygen consumption assay
1
Mitochondrial Respiration Assay in C2C12 Myotubes
2
Measuring Cellular Respiration Rates
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To determine respiration rates of HAP1 or HT29-MTX transfectants, consumption of extracellular oxygen was measured. Therefore, cells were seeded in a 96-well microtiter plate at a density of 1 × 105 cells per well and incubated at 37°C and 5% CO2 in a humidified incubator for 4 to 6 h. Real-time measurement of oxygen consumption was performed using the MitoXpress Xtra Oxygen Consumption Assay (Agilent) according to the manufacturer’s instructions.
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MitoXpress Xtra Oxygen Consumption Assay
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The OCR was determined using the commercial MitoXpress Xtra Oxygen Consumption Assay (Agilent) according to the manufacturer’s instructions. Fluorescence intensity (excitation light: 380 nm; measured light: 645 nm) was detected with the Synergy H1 microplate reader (BioTek, Winooski, USA) at 2-min intervals for 120 min, and subsequent conversion to lifetime values was performed using Gen5 software (BioTek).
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