hiPSC-fibs were seeded into vitronectin coated Seahorse 24 assay plates at a density of 80 000cells/well. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed using a standard mitochondrial and glycolysis stress on the Seahorse Bioscience XF-24 analyzer (Agilent technologies). On the day of metabolic flux analysis, cells were washed with Seahorse XF base medium pH7.4 (Agilent technologies). The culture medium was replaced with 500 µl of Seahorse medium supplemented with 10 mM glucose, 1 mM sodium pyruvate and 2 mM glutamine and incubated 1 h in a CO2-free incubator at 37 °C. For XF glycolysis stress test, cells were incubated in DMEM with 2 mM glutamine. For the XF mito stress, inhibitors of the mitochondrial electron transport chain (oligomycin 1 µM, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) 4 µM, rotenone and antimycin 1 µM) were sequentially injected to assess the OCR and the respiratory parameters. For XF glycolysis stress, glucose 10 mM, oligomycin 1 µM and 2-deoxy-D-glucose (2-DG) were injected to evaluate the ECAR and the glycolysis parameters. OCR and ECAR were automatically calculated by the Seahorse XF-24 software.
Seahorse xf base medium ph7
Manufactured by Agilent Technologies
Seahorse XF base medium pH7.4 is a specialized cell culture medium formulated for use with Agilent Seahorse XF Analyzers. It is designed to maintain a stable pH of 7.4 during the assay process. The medium provides a consistent, defined environment for cellular metabolism measurements.
Automatically generated - may contain errors
5 protocols using seahorse xf base medium ph7
1
Mitochondrial and Glycolytic Profiling of hiPSC-Fibroblasts
2
Seahorse Analysis of Mitochondrial Respiration
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Mitochondrial respiration was measured with Seahorse XFe24 Analyzer (Agilent Technologies) according to the manual of Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, cat#103015-100). Briefly, SCs isolated by FACS were plated on Matrigel-coated XF24 cell culture microplate at a density of 10,000 cells per well. Seahorse sensor cartridge was hydrated with calibrant in a non-CO2 incubator at 37°C for overnight 1 day before measurement. On the day of measurements, cells were washed twice and switched to Seahorse XF base medium (pH7.4, Agilent Technologies, cat#103334-100) supplemented with 1 mM sodium pyruvate, 1 mM l -glutamine, and 5 mM glucose. Cells were equilibrated at 37°C in a non-CO2 incubator for 1 hour. For FAO inhibition, Etomoxir (Sigma-Aldrich, cat#E1905) were added to cells at a final concentration of 50 μM 15 min before the measurement. The oxygen consumption rate was monitored at the basal state and after sequential injection of the mitochondrial compounds oligomycin (1.5 μM), FCCP (3 μM), and Rotenone/antimycin A (1 μM) to induce mitochondrial stress. All mitochondrial respiration rates were generated and automatically calculated by the Seahorse Wave software with normalization to the cellular protein contents.
3
Mitochondrial and Glycolytic Profiling of hiPSC-Fibroblasts
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hiPSC-fibs were seeded into vitronectin coated Seahorse 24 assay plates at a density of 80 000cells/well. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed using a standard mitochondrial and glycolysis stress on the Seahorse Bioscience XF-24 analyzer (Agilent technologies). On the day of metabolic flux analysis, cells were washed with Seahorse XF base medium pH7.4 (Agilent technologies). The culture medium was replaced with 500 µl of Seahorse medium supplemented with 10 mM glucose, 1 mM sodium pyruvate and 2 mM glutamine and incubated 1 h in a CO2-free incubator at 37 °C. For XF glycolysis stress test, cells were incubated in DMEM with 2 mM glutamine. For the XF mito stress, inhibitors of the mitochondrial electron transport chain (oligomycin 1 µM, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) 4 µM, rotenone and antimycin 1 µM) were sequentially injected to assess the OCR and the respiratory parameters. For XF glycolysis stress, glucose 10 mM, oligomycin 1 µM and 2-deoxy-D-glucose (2-DG) were injected to evaluate the ECAR and the glycolysis parameters. OCR and ECAR were automatically calculated by the Seahorse XF-24 software.
4
Mitochondrial and Glycolytic Profiling of hiPSC-Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hiPSC-fibs were seeded into vitronectin coated Seahorse 24 assay plates at a density of 80 000cells/well. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed using a standard mitochondrial and glycolysis stress on the Seahorse Bioscience XF-24 analyzer (Agilent technologies). On the day of metabolic flux analysis, cells were washed with Seahorse XF base medium pH7.4 (Agilent technologies). The culture medium was replaced with 500 µl of Seahorse medium supplemented with 10 mM glucose, 1 mM sodium pyruvate and 2 mM glutamine and incubated 1 h in a CO2-free incubator at 37 °C. For XF glycolysis stress test, cells were incubated in DMEM with 2 mM glutamine. For the XF mito stress, inhibitors of the mitochondrial electron transport chain (oligomycin 1 µM, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) 4 µM, rotenone and antimycin 1 µM) were sequentially injected to assess the OCR and the respiratory parameters. For XF glycolysis stress, glucose 10 mM, oligomycin 1 µM and 2-deoxy-D-glucose (2-DG) were injected to evaluate the ECAR and the glycolysis parameters. OCR and ECAR were automatically calculated by the Seahorse XF-24 software.
5
Seahorse Analysis of Mitochondrial Respiration
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