We measured the intracellular oxygen levels in the Mecp2+/− and CTRL MSCs to evaluate carbohydrates, lipids, and glutamine contribution to oxygen consumption. The MitoXpress® Xtra assay (Agilent Technologies, Luxcel Biosciences, Cork, Ireland) allowed us to determine extracellular oxygen consumption rates (OCR). The assay is based on the use of an oxygen-sensing fluorophore that is quenched by O2 through molecular collision. Therefore, the amount of fluorescence signal is inversely proportional to the amount of extracellular O2 in the analyzed samples. We first determined basal respiration. Subsequently, we added oligomycin and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), which target components of the electron transport chain (ETC) in the mitochondria to reveal key parameters of metabolic function. The addition of these components allowed us to determine oxygen employed for mitochondrial ATP production and maximal respiration, respectively. The OCR was determined according to the manufacturer’s guidance and instructions.
Mitoxpress xtra assay
Manufactured by Agilent Technologies
Sourced in Ireland
The MitoXpress® Xtra assay is a fluorescence-based tool designed to measure oxygen consumption in cells and mitochondria. It provides a non-invasive method for monitoring real-time changes in oxygen levels within samples.
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2 protocols using mitoxpress xtra assay
1
Evaluating Metabolic Profiles in Mecp2+/- MSCs
2
Quantifying Extracellular Oxygen Consumption
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We used the MitoXpressXtra assay (Agilent Technologies, Luxcel Biosciences) to determine extracellular oxygen consumption rates (OCR). Mouse FRCs were treated with control media or the conditioned media of 4T1 mouse breast cancer cells or MMTV-PyMT cancer cells isolated from mice for 48 h. After incubation, cells were washed with 1X PBS and the MitoXpress-Xtra-HS probe was added to cells in accordance with manufacturer’s instructions. Oxygen consumption was measured using time-resolved fluorescence (TR-F) with a dual delay of 30 μs and 70 μs using a CLARIOstar fluorescence plate reader (BMG Labtech).
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