OCR and ECAR were determined using XFp analyzer (Seahorse Bioscience, North Billerica, MA, USA). XFp cell mito-stress test kit, XFp glycolysis stress test kit, and XFp Real-Time ATP rate assay kit (Seahorse Bioscience) were used to detect OCR, ECAR, and ATP ratio, respectively. All the reagents and assay conditions were followed by manufacturer's instructions.
Xfp glycolysis stress test kit
Manufactured by Agilent Technologies
The XFp Glycolysis Stress Test Kit is a laboratory equipment product from Agilent Technologies. It is designed to measure and analyze cellular glycolysis and mitochondrial function in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Xfp cell mito stress test kit, by Agilent Technologies (3 mentions)
Xfp real time atp rate assay kit, by Agilent Technologies (1 mentions)
Xfp analyzer, by Agilent Technologies (1 mentions)
Seahorse xf24 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Base medium, by Agilent Technologies (1 mentions)
4 protocols using xfp glycolysis stress test kit
1
Cellular Bioenergetics Analysis with XFp
2
Mitochondrial and Glycolytic Profiling
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The OCR and ECAR were measured in XFp extracellular flux analyzers (EFAs) (Seahorse Bioscience) using a XFp Cell Mito Stress Test Kit and a XFp Glycolysis Stress Test kit, respectively. The following parameters were used in the assays: seed cells 8 × 104 per well, Oligomycin 1.0 μM, FCCP 1.0 μM, Rotenone/antimycin A 0.5 μM, glucose 10 mM, and 2-DG 50 mM as indicated.
3
Mitochondrial Metabolism Profiling of MSCs
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To evaluate the mitochondrial metabolism of the MSCs, the extracellular flux of the mitochondria was analyzed. Kinetic metabolic profiling was performed in real time using the fully integrated 8-well Seahorse Bioscience Extracellular Flux Analyzer XFp (Seahorse Bioscience, North Billerica, MA, USA). With this system, the XFp Cell Mito Stress Test Kit (Seahorse Bioscience) was used to determine the OCR as a surrogate for mitochondrial respiration, and the XFp Glycolysis Stress Test Kit (Seahorse Bioscience) was used to determine the extracellular acidification rate (ECAR) to measure the glycolytic reserve of the cells.
4
Analyzing Cellular Glycolytic Activity
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The extracellular acidification rates (ECAR) were measured using the Seahorse Extracellular Flux XF-24 analyzer (Seahorse Bioscience, N. Billerica, MA, USA). Cells were plated in XF24 cell culture microplates at a density of 8 × 104 cells/well in DMEM. After 24 h, the culture medium was changed to Base Medium (102353-100, Seahorse Bioscience, N. Billerica, MA, USA) supplemented with 2 mM glucose and was incubated at 37 °C for 1 h in a CO2-free incubator. The extracellular acidification rate (ECAR) was measured using the XFp Glycolysis Stress Test Kit (Seahorse Bioscience), according to the manufacturer’s instruction. Accordingly, after baseline measurement, the following injections were made—10 mM glucose, 1 μM oligomycin, and 50 mM 2-DG. ECAR values were normalized to the cell number. Cells were counted in a hemocytometer and viability was determined by trypan blue staining.
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