A Seahorse XFe24 Extracellular Flux Analyser (Seahorse Bioscience, Santa Clara, CA, US) was used to measure OCR as described elsewhere [40 (link)]. Primary pre-adipocytes were seeded on to 24-well Seahorse Mitcroplates coated with 0.1% gelatine, and were differentiated and treated as detailed above. Media was changed to Seahorse XF media one hour before undertaking the assays. The XFe Cell Mito Stress Test was carried out using 2 μM Oligomycin, 2 μM FCCP and 0.5 μM rotenone/antimycin (n = 5); preliminary experiments were used to determine optimal drug concentrations (data not shown). In a separate assay, isoproterenol was injected into the wells containing differentiated, treated primary adipocytes at a final concentration of 10 μM to determine their ability to respond to an adrenergic stimulus (n = 5). Values from both assays were normalised to total protein.
Seahorse xfe24 extracellular flux analyser
Manufactured by Agilent Technologies
Sourced in United States
The Seahorse XFe24 Extracellular Flux Analyser is a laboratory instrument designed to measure the metabolic activity of cells in real-time. The device utilizes sensor cartridges to simultaneously measure the oxygen consumption rate and extracellular acidification rate of cell samples, providing insights into cellular respiration and glycolysis.
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Lab products found in correlation
Xfe24 microplate, by Agilent Technologies (2 mentions)
Seahorse xf base dmem, by Agilent Technologies (1 mentions)
Glucose, by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Glycolysis stress test kit, by Agilent Technologies (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Glomax microplate reader, by Promega (1 mentions)
Mito stress test, by Agilent Technologies (1 mentions)
8 protocols using seahorse xfe24 extracellular flux analyser
1
Extracellular Flux Analysis of Adipocyte Metabolism
2
Measuring Glycolysis in CD4+ T Cells
3
Measurement of Mitochondrial Respiration in DRG Neurons
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DRG neuronal cells were cultured on V7 Seahorse 24-well cell culture microplates (Agilent Technologies), in maintenance medium in a 5% CO2 37 °C incubator. Sodium azide was present in media at either 0.1 or 1 mM for 17 h before the Seahorse experiment, and the sodium azide was maintained in the media during the Seahorse run. Plates were incubated for 30 min at 37 °C (without CO2), before entry into the Seahorse XFe24 Extracellular Flux Analyser (Agilent). Three measurements were taken basally, and three measurements taken after injection of each drug to either inhibit ATP-linked respiration, uncouple respiration or inhibit the respiratory chain. Mitochondrial respiration was calculated by subtracting the first OCR measurement following injection of antimycin/rotenone from the third basal OCR measurement. Normalisation of OCR to relative protein content was achieved following Sulforhodamine B (SRB) staining of all cell wells. Data for each treatment groups was averaged from between 4 and 5 replicate wells.
4
Metformin Effects on Cellular Respiration
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Cells were seeded at optimised seeding densities (HCT116: 10,000 cells/well, SW837: 30,000 cells/well in a final volume of 100 µL cRPMI) in a 24-well cell culture XFe24 microplate (Agilent Technologies, Santa Clara, CA, USA) and allowed to adhere at 37°C, 5% CO2/95% humidified air. At 5 h post seeding, an additional 150 µL complete media was added to each well. Following 24 h, medium was removed to waste, and cells were treated with metformin (2.5 or 10 mM) (Sigma Aldrich, St. Louis, MO, USA) or vehicle control (H2O) diluted in cRPMI. Following 24 h, treatment was removed and cells were washed with unbuffered Seahorse XF Base DMEM (Agilent) (supplemented with 10 mM glucose (Sigma), 10 mM sodium pyruvate (Sigma) and L-glutamine) and placed in a non-CO2 incubator for 1 h at 37°C. OCR and ECAR were measured using the Seahorse XFe24 Extracellular Flux Analyser (Agilent). Three baseline measurements of OCR and ECAR were taken over 24 min, consisting of 2 repetitions of mix (3 min)/wait (2 min)/measurement (3 min), to establish basal respiration. All OCR/ECAR readings were normalised using the crystal violet assay.
5
Measurement of Cellular Glycolysis
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Extracellular acidification rate (ECAR) was measured by using the Glycolysis Stress Test kit (Agilent Technologies, 103020‐100) according to the assay protocol on a Seahorse XFe24 Extracellular Flux Analyser (Agilent Technologies). Briefly, after washing the cells, they were resuspended in the culture medium supplemented with 2 mM glutamine, and incubated for 30 min without CO2. Glycolysis was measured by adding glucose (10 mM), oligomycin (1.5 μM) and 2‐deoxy‐D‐glucose (50 mM).
6
Metabolic Activity of Expanded NK Cells
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Expanded NK cells were seeded at a density of 400,000 cells per 100 ul in a well of a 24-well cell culture XFe24 microplates (Agilent Technologies) pre-coated with poly-d -lysine (Sigma). Cells were treated with obese or non-obese ACM or TCM for 24 h. Prior to the commencement of the assay microplates were centrifuged at 1300 rpm for 3 min to allow for adherence. Media was removed and cells were washed with unbuffered Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10 mM of glucose and 10 mM of sodium pyruvate, (pH 7.4) and incubated for one hour at 37 °C in a CO2-free incubator. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe24 Extracellular Flux Analyser (Agilent Technologies).
Measurements were normalised to cell number using the crystal violet assay. Cells were fixed with 1% glutaraldehyde for 15 min. The fixative was removed, and cells were washed with PBS and stained with 0.1% crystal violet in PBS for 30 min. Plates were left to air dry overnight and incubated with 50 μl 1% Triton X-100 in PBS on a plate shaker for 30 min. Absorbance was read at 595 nm on a GloMax microplate reader (Promega).
Measurements were normalised to cell number using the crystal violet assay. Cells were fixed with 1% glutaraldehyde for 15 min. The fixative was removed, and cells were washed with PBS and stained with 0.1% crystal violet in PBS for 30 min. Plates were left to air dry overnight and incubated with 50 μl 1% Triton X-100 in PBS on a plate shaker for 30 min. Absorbance was read at 595 nm on a GloMax microplate reader (Promega).
7
Platelets Bioenergetic Profile Analysis
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Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe24 Extracellular Flux Analyser (Agilent Technologies, Stockport, UK). Washed platelets (2.5 x 10 7 cells/well) were resuspended in DMEM assay medium. Platelets were kept at 37 ᵒC for 30 minutes with 30 µM CxxCpep, 30 µM Scrambled peptide (VGCPAKWCYHEF, synthesized by PeptideSynthetics, Fareham, UK) or vehicle and loaded into the Seahorse XFe24 Extracellular Flux Analyser. Baseline measurements of OCR and ECAR were performed at the beginning of the assay, followed by the addition of thrombin or collagen, oligomycin to inhibit ATP synthase, cyanide p-trifluoro-methoxy phenyl-hydrazone (FCCP) to uncouple oxidative phosphorylation and inhibitors of complex I and III rotenone and antimycin A, respectively. Respiratory parameters were calculated according to [22] [23] [24] .
8
Mitochondrial Bioenergetics Profiling of Cells
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A mitochondrial bioenergetics analysis was performed by measuring the oxygen consumption rate (OCR) of cultured cells using the Seahorse XFe24 Extracellular Flux analyser (Agilent, Santa Clara, CA, USA). Cells were plated at 30,000 cells/ well and left overnight to adhere. Cells were then treated with Aze at 2000 µM for 24 h. The Mito Stress Test (Agilent) was then performed according to manufacturer's instructions. Cells were then washed in Seahorse assay media and prepared using the manufacturer's protocol and kit reagents (oligomycin (1 µM), FCCP (1 µM) and antimycin A/rotenone (0.5 µM)). OCR values were normalised to protein concentration using the BCA assay. Basal respiration, maximal respiration, ATP production and non-mitochondrial respiration were then calculated using the Seahorse XF Cell Mito Stress Test Report Generator. Calculations are as follows; non-mitochondrial respiration equates to the minimum rate measurement after antimycin A/rotenone injection, basal respiration equates to last rate measurement before oligomycin injection subtracted from non-mitochondrial respiration, maximal respiration equates to maximum rate measurement after FCCP injection subtracted from non-mitochondrial respiration and ATP production equates to last rate measurement before oligomycin injection subtracted from the minimum rate after oligomycin injection.
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