SW837 cells were seeded in duplicate at a density of 30,000 cells per well in complete RPMI in a 24 well XFe24 cell culture microplate for 24 h [Agilent Technologies, Santa Clara, CA, USA] and then treated with NCM or TCM from the human normal or rectal cancer ex vivo explants respectively for 24 h. Following 24-h treatment, Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were measured using a Seahorse Biosciences XFe24 Extracellular Flux Analyser [Agilent Technologies, Santa Clara, CA, USA] as previously described [15 ].
Xfe24 extracellular flux analyser
Manufactured by Agilent Technologies
Sourced in United States
The XFe24 extracellular flux analyser is a lab equipment product from Agilent Technologies. It is designed to measure the extracellular flux of cells in real-time, providing data on cellular oxygen consumption and extracellular acidification rates.
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Lab products found in correlation
Xfe24 cell culture microplate, by Agilent Technologies (1 mentions)
Seahorse xf24 cell culture microplate, by Agilent Technologies (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
Pierce rapid gold bca protein assay, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Mito stress test kit, by Agilent Technologies (1 mentions)
Stellux chemi human c peptide elisa kit, by ALPCO (1 mentions)
Xf cell mito stress test generator, by Agilent Technologies (1 mentions)
Wave software, by Agilent Technologies (1 mentions)
Xf base medium, by Agilent Technologies (1 mentions)
Palmitate, by Merck Group (1 mentions)
Rotenone antimycin a, by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (1 mentions)
Cell tak, by Corning (1 mentions)
11 protocols using xfe24 extracellular flux analyser
1
Measuring Metabolic Responses in Cancer Cells
2
Assessing Caco-2 Cells' Metabolic Profiles
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Caco-2 cells (2.0x104 cells/well) were cultured in Seahorse XF24 cell culture microplates (Agilent, Santa Clara, CA, USA) by using DMEM-galactose for 15 days. Differentiated cells were then incubated with A. simplex CE (0.5, 1 and 2mg/mL) for two hours. Thereafter, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured with the XFe24 Extracellular Flux Analyser (Agilent) from the Cell Culture Service of Centro de Investigaciones Biológicas (CIB-CSIC). Basal OCR and ECAR were determined first. Then leak OCR and maximal ECAR were measured with 2μM oligomycin, maximal OCR with 2μM and 3μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, Sigma-Aldrich) and non-mitochondrial OCR with 2.5μM antimycin A and 1.25μM rotenone. The basal, leak, and maximal OCR values were corrected for non-mitochondrial OCR. OCR and ECAR values were normalized by cellular protein content as was analysed with the Pierce Rapid Gold BCA Protein Assay (Thermo Fisher Scientific). Four experiments in five replicates were performed.
3
Measuring Cellular Oxygen Consumption
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Oxygen consumption rates (OCR) were measured using the XFe24 Extracellular Flux Analyser (AgilentTechnologies, Les Ulis, France). Briefly, culture medium was removed and cells were washed once with DMEM (Sigma-Aldrich), pH buffered at 7.35, supplemented with 10 mM glucose and 2 mM glutamine. Then, cells were incubated with DMEM in CO2-free chamber at 37 °C for 30 min. Baseline OCR was measured: (i) at the basal state, (ii) after oligomycin (Oli) injection (1 μM), (iii) after FCCP injection (0.27 μM and then 0.34 μM), and (iv) after rotenone (Rot)/antimycin A (AA) mix injection (1 μM).
4
Cellular Oxygen Consumption Profiling
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Cellular oxygen consumption rate (OCR) was measured using an XFe24 Extracellular Flux Analyser (Agilent Seahorse, Billerica, MA, USA) and the Mito Stress Test Kit, according to the manufacturer's protocol. H9c2 cells (2 × 105) were plated in 100 μL of their standard growing media and cultured overnight. On the day of measurement, cells were washed with XFe24 media and incubated in a CO2-free incubator at 37 °C for 2 h to establish equilibration prior to loading. OCR measurements were obtained before and after addition of glucose (10 mmol/L), oligomycin (1 μmol/L), FCCP (4 μmol/L), and rotenone/antimycin A (0.5 μmol/L). These measurements were used to calculate basal respiration, ATP production, and maximal respiration.
5
Mitochondrial Respiration Profiling of hPSC-Derived Clusters and Human Islets
6
Analyzing Endothelial Cell Respiration
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An XFe24 extracellular flux analyser (Seahorse Biosciences, MA) was used to measure oxygen consumption rates [23 (link)]. Briefly, thirty thousand of treated endothelial cells were seeded in a provided 24-well plate in the CO2 incubator. One hour before measurements, cells were removed and incubated at 37 °C in a normal atmosphere. The medium was then replaced with FX assay medium. Stock solutions of oligomycin (1 μM at final concentration), FCCP (0.5 μM at final concentration) and rotenone/antimycin A (0.5 μM/0.5 μM at final concentration) in an XF Cell Mito Stress Test Kit were prepared in FX assay medium and loaded into indicated injection ports respectively. Measurements were obtained at 37 °C. Assay cycles included 3 min of mixing, 2 min of waiting period, and 3 min of measurement. The Wave software provided by Seahorse Biosciences was used for data analysis. Basal respiration (Mean of Stage A – Mean of Non-mitochondria respiration (Stage D)), maximal respiration (Mean of Stage C – Mean of Non-mitochondria respiration (Stage D)) and ATP production (Mean of Stage A – (Mean of Non-mitochondria respiration+proton leak) (Stage B)) were determined by the XF Cell Mito Stress Test Generator that was provided by Seahorse Bioscience.
7
Measurement of Cellular Metabolic Flux
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As described previously (Liu et al., 2017 (link); Yang et al., 2018 (link)), cells were seeded onto Seahorse XF24 polystyrene tissue culture plates (Seahorse Bioscience) at a density of 5 × 104 cells per well and incubated at 37°C overnight. The next day, the medium was changed to XF Base Medium (Seahorse Bioscience) supplemented with 25‐mM glucose, 2‐mM glutamine and 1‐mM pyruvate, pH adjusted to 7.4 with 0.1‐M NaOH, and then the plate was incubated in a non‐CO2 incubator at 37°C for 1 h. The extracellular acidification rate (ECAR) was measured with an XFe24 extracellular flux analyser (Seahorse Bioscience). Inhibitors and activators were used at the following concentrations: glucose (10 mM), oligomycin (2 μM) and 2‐DG (50 mM).
8
Mitochondrial Fuel Flexibility Assay
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ECAR and OCR were measured using an XFe24 extracellular flux analyser (Seahorse Bioscience; North Billerica, MA). Briefly, cells were seeded at 4×104 per well in 525 μL of XF assay medium supplemented with 2 mM glutamine (for ECAR) or 2 mM glutamine, 10 mM glucose, and 1 mM pyruvate (for OCR). Cells were incubated in a CO2-free incubator for 1 h at 37°C to allow for temperature and pH equilibration prior to loading into the XFe24 analyser for measurement following the manufacturer’s protocol. Basal glycolysis was calculated by subtracting the third baseline (non-glycolytic acidification) ECAR reading from the third glycolysis ECAR reading following glucose addition. Basal respiration was derived by subtracting the third OCR reading following antimycin A addition from the third basal OCR reading. For Mito Fuel Flex Test, inhibitors of glucose (UK5099), glutamine (BPTES), and fatty acid (etomoxir) pathways were applied following the manufacturer’s protocol. Fuel dependency is tested by first injecting an inhibitor of the target pathway, followed by inhibition of the two alternative pathways. Fuel capacity is tested by first injecting inhibitors of the alternative pathways, followed by inhibition of the target pathway. Fuel flexibility is calculated as the difference between capacity and dependency.
9
Mitochondrial Fuel Flexibility Assay
10
Cellular Oxygen Consumption Assay
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Cellular OCR was measured using the XFe24 Extracellular Flux Analyser (Seahorse Bioscience, USA). SVFs isolated from male 6-week-old A-FABP KO mice and their relative WT littermates were seeded in XFe24-well microplate with 1.5 × 104 cells per well and differentiated into mature adipocytes. Cells were pre-incubated with BSA (3 μg ml−1), rA-FABP (2 μg ml−1) or palmitate (200 nM, Sigma-Aldrich) for 30 min. Oligomycin (5 μM, ATP synthase inhibitor, Sigma-Aldrich), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 50 μM, cellular uncoupler, Sigma-Aldrich), rotenone/antimycin A (1 μM, Sigma-Aldrich) were sequentially added to determine basal-, ATP-dependent-, maximal- and mitochondria-independent oxygen consumption, respectively.
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