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 bioscience xf 24 analyzer
Manufactured by Agilent Technologies
Sourced in Japan, United States
The Seahorse Bioscience XF-24 analyzer is a lab equipment product that measures the oxygen consumption rate and extracellular acidification rate of cells in real-time. It provides insights into cellular metabolism and bioenergetics.
Automatically generated - may contain errors
Lab products found in correlation
Seahorse xf base medium ph7, by Agilent Technologies (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Xf24 cell culture microplate, by Agilent Technologies (2 mentions)
Minimal dmem, by Merck Group (2 mentions)
Glucose, by Junsei (2 mentions)
Oligomycin, by Merck Group (2 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rotenone, by Merck Group (2 mentions)
Pyruvate, by Merck Group (1 mentions)
Glutamax, by Merck Group (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Xf24 calibrant solution, by Agilent Technologies (1 mentions)
Cyanide 3 chlorophenylhydrazone cccp, by Merck Group (1 mentions)
7 protocols using seahorse bioscience xf 24 analyzer
1
Mitochondrial and Glycolytic Profiling of hiPSC-Fibroblasts
2
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.
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
Measuring Metabolic Profiles of Colonic MSCs
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Primary cultured colonic MSCs were seeded at 4 × 104 cells per well in XF24 cell culture microplates (Agilent Technologies) and cultured in MSC culture medium overnight. One hour before measurement, culture medium was replaced with OCR assay medium (minimal DMEM [Sigma-Aldrich] and supplemented with 20 mmol/L glucose [Junsei Chemical, Tokyo, Japan], 2 mmol/L GlutaMax [Gibco], and 5 mmol/L pyruvate [Sigma-Aldrich]) or ECAR assay medium (minimal DMEM and supplemented with 2 mmol/L GlutaMax) in the 37°C non-CO2 incubator. The Seahorse Bioscience XF24 analyzer (Agilent Technologies) was used to measure OCR and ECAR. For OCR measurements, 1 μmol/L oligomycin (Sigma-Aldrich), 1 μmol/L FCCP (Sigma-Aldrich), and 1 μmol/L rotenone and antimycin (Sigma-Aldrich) were injected.. For ECAR measurements, 10 mmol/L glucose, 2 μmol/L oligomycin, and 50 mmol/L 2-deoxy-D-glucose (Sigma-Aldrich) were injected. The parameters of glycolytic function, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification were calculated using Seahorse Wave software V2.6.
5
Organoid Oxygen Consumption Measurement
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Organoids were dissociated with TrypLE Express for 10 min at 37 °C. Dissociated single cells were seeded at 5 × 103 cells per well with Matrigel in XF24 cell culture microplates (Agilent Technologies) and cultured in EN medium for 5–6 days. One hour before measurement, culture medium was replaced with OCR assay media [minimal DMEM (Sigma Aldrich) supplemented with GlutaMAX (2 mM, Thermo Fisher), pyruvate (5 mM, Thermo Fisher), glucose (20 mM, Junsei Chemical, Tokyo, Japan)] in the 37 °C non-CO2 incubator for 1 h. We used a Seahorse Bioscience XF24 analyzer (Agilent Technologies) to measure OCR. Oligomycin (1 μM), FCCP (1 μM), and rotenone and antimycin (1 μM) were injected for OCR measurements. All reagents were purchased from Sigma Aldrich. After the measurements, cells were lysed with RIPA buffer (Thermo Fisher) and isolated proteins were quantified with Pierce BCA Protein Assay Kit (Thermo Fisher) for normalization.
6
Mitochondrial Respiration Profiling in Macrophages
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The XF24 biosensor cartridge was activated with 1 ml of XF24 calibrant solution (Seahorse Bioscience, Billerica, MA) per well for 24 h at 37°C in a non-CO2 incubation system. SIRT3 WT and KO BMDMs infected with Mabc for 18 h were seeded at 2 × 104 cells per well and incubated for 24 h at 37°C. The cell plate was incubated for 1 h at 37°C in a non-CO2 incubation system after the addition of 590 µl assay media in each well. ATPase inhibitor oligomycin A (20 µg/mL, Sigma-Aldrich, MO, USA), uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 50 µM, Sigma-Aldrich, MO, USA), and mitochondrial complex I inhibitor rotenone (20 μM, Sigma-Aldrich, MO, USA) were sequentially added to each well after measurement of basal OCR. Seahorse Bioscience XF24 analyzer (Seahorse Bioscience, Billerica, MA) was used to measure the oxygen consumption rate of the entire process.
7
Mitochondrial Respiration Assay in Cells
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Mitochondrial oxygen consumption in intact cells were measured with the Seahorse Bioscience XF-24 analyzer (Seahorse Bioscience, Billerica, MA, USA) and reported as oxygen consumption rate (OCR) as described previously (Brand & Nicholls 2011 (link)). HepIR cells were seeded at 15 000 cells/well into XF-24 culture microplates and cultured overnight at 37°C with 5% CO2. During the 24 h post-treatment with or without TM, the medium was replaced with pre-warmed 600 μl of sodium carbonate-free DMEM for 1 h. Each experimental condition was analyzed using four to six biological replicates. The following reagents including 1 μM oligomycin (Sigma-Aldrich), 1 μM FCCP (Sigma-Aldrich) or 1 μM rotenone (Sigma-Aldrich) were added to block state III respiration, induce uncoupling, or shut down mitochondrial respiration, respectively. Data were normalized to protein content.
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