For real-time analysis of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of purified and expanded NK cells cultured under various conditions, a Seahorse XF-24 Analyser or a Seahorse XFe-96e Analyser (Agilent Technologies) was used. In brief, 750,000 MACS purified, expanded NK cells were added to a 24-well XF Cell Culture Microplate, 200,000 MACS purified NK cells to a 96-well XFe Cell Culture Microplate. All cell culture plates were treated with Cell-Tak (BD Pharmingen) to ensure that the NK cells adhere to the plate. Sequential measurements of ECAR and OCR following addition of the inhibitors (Sigma) oligomycin (2 μM), FCCP (1 μM), rotenone (100 nM) plus antimycin A (4 μM), and 2-deoxyglucose (2DG, 30 mM) allowed for the calculation of basal glycolysis, glycolytic capacity, basal mitochondrial respiration, and maximal mitochondrial respiration.
Seahorse xf24 analyser
Manufactured by Agilent Technologies
Sourced in United States
The Seahorse XF24 Analyzer is a laboratory instrument designed to measure the metabolic activity of cells in real-time. It provides simultaneous measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are indicators of cellular respiration and glycolysis, respectively.
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Lab products found in correlation
Seahorse xf cell mito stress test kit, by Agilent Technologies (2 mentions)
Cell tak, by BD (2 mentions)
Seahorse assay medium, by Agilent Technologies (1 mentions)
Glycolysis stress test kit, by Agilent Technologies (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Bms 303141, by Merck Group (1 mentions)
Seahorse xfe96 analyser, by Agilent Technologies (1 mentions)
Sb204990, by Bio-Techne (1 mentions)
Xf cell culture microplate, by Agilent Technologies (1 mentions)
Bptes, by Bio-Techne (1 mentions)
Oligomycin, by Agilent Technologies (1 mentions)
Antimycin a, by Agilent Technologies (1 mentions)
24 well xf microplate, by Agilent Technologies (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
Oligomycin a, by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Xf cell mito stress test kit, by Agilent Technologies (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Xf24 plate, by Agilent Technologies (1 mentions)
Xf24 analyser, by Agilent Technologies (1 mentions)
13 protocols using seahorse xf24 analyser
1
Metabolic Profiling of NK Cells
2
Cellular Metabolism Profiling by Seahorse
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Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), reflecting OXPHOS and glycolysis, respectively, were measured before and after treatment with oligomycin (2 μg/ml), trifluorocarbonylcyanide phenylhydrazone (FCCP; 5 μM), antimycin A (2 μM) and 2-deoxyglucose (2-DG; 25 mM) using the Seahorse XF24 analyser (Agilent Technologies, Santa Clara, CA, USA). RASFC and HUVEC were seeded at 30,000 cells per well in a Seahorse XF96 cell culture microplate (Agilent Technologies) and allowed to adhere for 24 hours. Cells were rinsed with assay medium (unbuffered DMEM supplemented with 10 mM glucose, 1 mM sodium pyruvate and 2 mM l -glutamine, pH 7.4) before incubation with assay medium for 30 minutes at 37 °C in a non-CO2 incubator. Following incubation, cells were stimulated with 4-HNE (2.5 μM) and vehicle basal medium for 2 hours. Four baseline OCR and ECAR measurements were obtained over 28 minutes before injection of specific metabolic inhibitors. Moreover, to challenge the metabolic capacity of the RASFC and HUVEC, three OCR and ECAR measurements were obtained over 15 minutes following injection with oligomycin, FCCP, antimycin A and 2-DG.
3
Seahorse XF Cell Mito Stress Test
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Hepatocytes were seeded into XF24 cell culture plates. After drug treatment (FFA or BBR), the media were removed, and cells were washed twice with warm Seahorse assay medium (Agilent Technologies, USA) and incubated for 1 h at 37 °C without CO2. The compounds oligomycin, FCCP, antimycin, and rotenone of the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, USA) were diluted in Seahorse assay medium and were preloaded into reagent delivery ports of A, B, and C of the O2 sensor cartridge. Oxygen consumption rate (OCR) measurements were then carried out according to the Seahorse assay protocol using a Seahorse XF-24 analyser (Agilent technologies, USA). The basal level of OCR was measured 4 times, and the maximal respiratory capacity was determined by OCR after FCCP injection, minus the one before oligomycin injection.
4
Glycolysis Measurement via Seahorse XF24
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Experiments for measuring cellular glycolysis were performed using a Seahorse XF24 Analyser (Agilent, Santa Clara, CA, USA) and Glycolysis Stress Test kit (Agilent, Santa Clara, CA, USA). The optimal cell seeding number was determined by initial titration experiments. The Seahorse sensor cartridge was hydrated for 24 h at 37 °C in XF Calibrant liquid prior to each experiment. Immediately before the experiment, each plate well was loaded with 80 µL of the supplied Glycolysis kit compounds (10 mM glucose, 2.5 µM oligomycin, and 100 mM 2-Deoxy-D-Glucose), according to the manufacturer’s specifications. After seeding 80,000 cells/well, the cells were allowed to adhere to the plate for 3 h before loading both the sensor plate and the cell culture plate into the Seahorse machine for analysis. The conditions comprised of an initial equilibration step (3 cycles of: 3 min—mix, 2 min—wait, 3 min—measure), the addition of a glucose step (3 cycles of: 3 min—mix, 2 min—wait, 3 min—measure), the addition of an oligomycin step (3 cycles of: 3 min—mix, 2 min—wait, 3 min—measure), and finally, the addition of 2-DG (3 cycles of: 3 min—mix, 2 min—wait, 3 min—measure). At the completion of the experiment, the data from each sample was analysed using the “Wave v2.3” software.
5
Monocyte Mitochondrial and Glycolytic Assay
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Monocytes were adhered to CellTak-coated 24-well plates at a concentration of 1 × 106 per well as described above. Cells were stimulated with 5 μg/ml anti-MPO, anti-PR3, isotype or vehicle for 4 h at 37°C with 5% CO2. Mitochondrial stress test or glycolysis stress test were performed as per the manufacturer's instructions using a Seahorse XF24 analyser (Agilent). Sequential addition of oligomycin (2 μM), D-glucose (5.5 mM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (4 μM), rotenone (4 μM), 2 deoxy-glucose (2-DG) (10 mM) allows for accurate calculation of mitochondrial respiratory capacity and glycolytic acidification. Each condition was evaluated in duplicate and a minimum of three measurements was performed following addition of each compound.
6
Mitochondrial Function Evaluation in 3T3-L1 Cells
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Mitochondrial function was assessed and analysed in live cells by Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies #103015), according to the manufacturer’s protocol. Briefly, 2×104 cells/well of 3T3-L1 cells were plated on a Seahorse XF24 cell culture microplate and transfected with siHif3α. At 24 h, the medium was replaced with differentiated medium in presence of pro-inflammatory cytokines. After 72 h, the plate was loaded into the by Seahorse XF24 Analyser (Agilent Technologies) and analysed as reported in (Carafa et al., 2020 (link)). Multiple parameters are evaluated including basal respiration, ATP-linked respiration, maximal and reserve capacities, and non-mitochondrial respiration.
7
Metabolic Profiling of Expanded NK Cells
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For real-time analysis of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of purified and expanded NK cells cultured under various conditions, a Seahorse XF-24 Analyser, a Seahorse XFe-96 Analyser or a Seahorse XF-8 Analyser (Seahorse Bioscience) was used. In brief, 500,000 to 750,000 MACS purified, expanded NK cells were added to a 24-well XF Cell Culture Microplate, 100,000 to 200,000 MACS purified NK cells to a 96-well XFe Cell Culture Microplate and 200,000 ex vivo pure NK cells to an 8-well XF Cell Culture Microplate (Seahorse Biosciences). All cell culture plates were treated with Cell-Tak™ (BD Pharmingen) to ensure that the NK cells adhere to the plate. Sequential measurements of ECAR and OCR following addition of the inhibitors (Sigma) oligomycin (2 μM), FCCP (1 μM), rotenone (100 nM) plus antimycin A (4 μM), and 2-deoxyglucose (2DG, 30 mM) allowed for the calculation of basal glycolysis, glycolytic capacity, basal mitochondrial respiration, and maximal mitochondrial respiration. Where indicated, BMS303141 (10 μM, Sigma), SB204990 (30 μM, Tocris), BPTES (10 μM,Tocris) or an equivalent amount of vehicle control was injected into the Seahorse plate.
8
Profiling Cellular Metabolism in Rheumatoid Arthritis
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Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), reflecting oxidative phosphorylation and glycolysis, respectively, were measured before and after treatment with oligomycin (2 µg/mL, Seahorse Biosciences, UK), trifluorocarbonylcyanide phenylhydrazone (FCCP) (5 μM, Seahorse Biosciences) and antimycin A (2 μM, Seahorse Biosciences) using the Seahorse XF24-analyser (Seahorse Biosciences). Rheumatoid arthritis synovial fibroblasts (RASF) were seeded at 30 000 cells per well in 24-well XF-microplates (Seahorse Biosciences) and allowed to adhere for 24 h. Cells were rinsed with assay medium (unbuffered Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10 mM glucose, pH7.4) before incubation with assay medium for 30 min at 37°C in a non-CO2 incubator. Following incubation cells were stimulated with dimethyloxalylglycine (DMOG), a pan prolyl hydroxylase inhibitor and dimethyl sulfoxide (DMSO) vehicle control for 2 h. DMOG (HIF1α activator), was used in these experiments to mimic the effect of hypoxia as the Seahorse Analyzer cannot be placed in a hypoxic chamber. Four baseline OCR and ECAR measurements were obtained over 28 min before injection of specific metabolic inhibitors. Moreover, to challenge the metabolic capacity of the RASF, three OCR and ECAR measurements were obtained over 15 min following injection with oligomycin, FCCP and antimycin A.
9
Bioenergetic Profiling of Cell Lines
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The ECAR and OCR were measured using a Seahorse XF24 analyser (Seahorse Bioscience, Boston, MA) according to the manufacturer’s instructions. Plate BV-2 cells at 8 × 104 cells/100 μL and PC-12 cells at 6 × 104 cells/100 μL previously in the Seahorse XF Microplate, respectively, and treated with vehicle, Sal (1, 10 and 100 μM) or DFO (100 μM). The sensor cartridge was hydrated in a Seahorse XF Calibrant and incubated overnight (37 °C, CO2-free). For ECAR measurements, the assay medium of PC-12 was prepared by XF base medium with 4 mM glutamine, and BV-2 was prepared by XF base medium with 2.5 mM glutamine. For OCR measurements, the assay medium of BV-2 (1 mM pyruvate, 2.5 mM glutamine and 17 mM glucose) and PC-12 (XF base medium containing 1 mM pyruvate, 4 mM glutamine and 25 mM glucose) were prepared immediately before assay.
10
Mitochondrial Respiration Profiling
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Oxygen consumption rate (OCR) was detected using a Seahorse XF24 analyser (Seahorse Bioscience), per the manufacturer’s instructions. Briefly, miR-155+/KO or miR-155KO/KOcells were cultured to approximately 70–80% confluence in DMEM medium. Cells were seeded at 50,000 cells per well in an XF24 cell culture microplate and then incubated at 37 °C with 5% CO2 for 3 h. Before OCR detection, culture medium was changed, and the plate was incubated at 37 °C without CO2 for 1 h. Finally, the plate was transferred to the XF24 analyser. OCR was measured by sequential additions of oligomycin A (Sigma), FCCP (Sigma) and antimycin A (Sigma).
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