Xf96e analyzer

Manufactured by Agilent Technologies

The XF96e Analyzer is a laboratory instrument designed for cellular bioenergetics analysis. It measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells in a 96-well microplate format, providing insights into cellular metabolism. The XF96e Analyzer enables researchers to study various cellular processes, including mitochondrial function, glycolysis, and metabolic profiling.

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Lab products found in correlation

Dctm protein assay kit 2, by Bio-Rad (1 mentions) Xf96 v3 ps cell culture microplate, by Agilent Technologies (1 mentions) Cyquant dye, by Thermo Fisher Scientific (1 mentions) L carnitine, by Merck Group (1 mentions) D glucose, by Merck Group (1 mentions) L glutamine, by Agilent Technologies (1 mentions) Hoechst solution, by Merck Group (1 mentions) Dmem high glucose medium, by Biowest (1 mentions) Wave software version 2, by Agilent Technologies (1 mentions)

Spelling variants of this product

XF96e Extracellular Flux Analyzer (26 citations) Seahorse XF96e analyzer (11 citations) XF96e (7 citations) Seahorse XF96e Extracellular Flux Analyzer (6 citations) Agilent Seahorse XF96e Analyzer (5 citations) XF96e Flux analyzer (2 citations)

7 protocols using xf96e analyzer

Cellular Respiration Analysis Using Seahorse

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Measurement of cellular respiration was performed using the Seahorse XF96e analyzer as previously described³². Cells were grown in either KGM Gold or RM + medium and seeded out as described above. Prior to respiration assays, cell culture medium was replaced by prewarmed, pH 7.4 adjusted assay medium supplemented with 5 mM glucose, 10 mM sodium pyruvate and 2 mM glutamate according to the manufacturer’s protocol. Oxygen consumption rate (OCR), extracellular consumption rate (ECAR) and proton production rate were measured under basal conditions and in the presence of oligomycin, a complex V inhibitor (1 μM), the complex III inhibitor antimycin A (0.5 μM), the complex I inhibitor rotenone (0.5 μM) and the mitochondrial uncoupler carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1.5 μM) to assess maximal oxidative capacity. Following the respiration assay, media was removed, wells were washed once with PBS and total protein concentration was measured using the DCTM Protein Assay Kit II (BioRad). All cell lines were at least measured n = 10 times and normalized to total protein content, including a BSA standard with known protein concentrations. The data was later analysed using the Seahorse Report Generator. Spare respiratory capacity (SRC) was defined as the difference between basal and maximum respiration.

Kirschberg M., Heuser S., Marcuzzi G.P., Hufbauer M., Seeger J.M., Đukić A., Tomaić V., Majewski S., Wagner S., Wittekindt C., Würdemann N., Klussmann J.P., Quaas A., Kashkar H, & Akgül B. (2020). ATP synthase modulation leads to an increase of spare respiratory capacity in HPV associated cancers. Scientific Reports, 10, 17339.

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Metabolic Profiling of Primary Epithelial Cells

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Metabolic profiling of primary epithelial cells was performed using XF96e analyzer (Seahorse Bioscience). Detailed methods are described in Supplemental Experimental Procedures.

Kaiko G.E., Ryu S.H., Koues O.I., Collins P.L., Solnica-Krezel L., Pearce E.J., Pearce E.L., Oltz E.M, & Stappenbeck T.S. (2016). The colonic crypt protects stem cells from microbiota-derived metabolites. Cell, 165(7), 1708-1720.

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Mitochondrial Respiration in SCO2 Cells

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For mitochondrial respiration studies using the Seahorse XF96e Analyzer, 1.5 × 10⁴ HCT116 SCO2^+/+, SCO2^−/−, and SCO2^−/−Tg cells from three different thawing batches were plated in Seahorse XF96 V3 PS Cell Culture Microplates and incubated for 16 h at standard conditions. XFe96 Sensor Cartridge was hydrated with ultrapure water overnight and, 45 min to 1 h prior to the commencement of the experiment, the cartridge was incubated with XF Calibrant solution at 37 °C in CO₂-free humidified incubator. Cell-containing plate was washed with XF Assay medium, pH 7.4, supplemented with d-glucose 3 g/l, pyruvate 1 mM, and l-glutamine 1 mM at 37 °C according to the manufacturer’s recommended protocol. Injection ports were loaded with 10× concentrations of the stock agents listed in the aforementioned “Mitochondrial modulator treatment” section. Appropriates volumes of 10× stock were added to a starting medium volume of 180 μl containing the cells.

Mori M.P., Penjweini R., Ma J., Alspaugh G., Andreoni A., Kim Y.C., Wang P.Y., Knutson J.R, & Hwang P.M. (2023). Mitochondrial respiration reduces exposure of the nucleus to oxygen. The Journal of Biological Chemistry, 299(3), 103018.

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Seahorse XF Analysis of Neutrophil and Splenocyte Metabolism

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Bone marrow derived neutrophils or splenocytes were plated on Cell-Tak coated Seahorse culture plates (300.000 cells/well) in Seahorse XF RPMI medium (pH 7.4). XF analysis was performed at 37°C with no CO2 using the XF-96e analyzer (Agilent) as per manufacturer’s instructions. Mitochondrial stress test assay was performed using Seahorse XF RPMI medium (pH 7.4) supplemented with 25 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine. Cells were treated serially with oligomycin (5 μM), FCCP (1 μM), and rotenone (100 nM) and antimycin A (10 uM), and oxygen consumption rates (OCR) were measured over time. The cell numbers at assay completion were normalized to DNA content using CyQuant dye (Thermofischer). Wave, Excel, and Graph Pad Prism software were used to analyze the data. Mann-Whitney U test was performed to calculate significance.

Blanco L.P., Pedersen H.L., Wang X., Lightfoot Y.L., Seto N., Carmona-Rivera C., Yu Z.X., Hoffmann V., Yuen P.S, & Kaplan M.J. (2020). The coenzyme Q10 analog idebenone attenuates murine lupus by improving mitochondrial metabolism and reducing inflammation.. Arthritis & rheumatology (Hoboken, N.J.), 72(3), 454-464.

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Metabolic Profile Characterization of Macrophages

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An XF96e Analyzer (Agilent Technologies, Santa Clara, CA) was used to measure bioenergetic function in isolated peritoneal macrophages. Prior to the run, sorted peritoneal macrophages were plated at a concentration of 0.6×10⁵cells/well. Cells were stimulated with MLE-12 or LLC exosomes (40 μg/mL) overnight. For all bioenergetic measurements, the culture media was changed 1 h prior to the assay run to unbuffered Dulbecco’s Modified Eagle Medium (DMEM, pH 7.4) supplemented with 11 mM D-Glucose (Sigma cat. G7528), 2 mM L-glutamine (Mediatech cat. 61-030-RM), and 100 μM L-carnitine (Sigma cat. C0283). The final concentrations of oligomycin (port A), FCCP (port B), and antimycin A/rotenone (port C) were 2 μg/ml, 2.5 μM and 10 μM/1 μM, respectively. Three basal OCR measurements were recorded prior to injection of oligomycin. After recording the oligomycin-sensitive OCR, FCCP-sensitive rates were recorded. Finally, antimycin A/rotenone was injected to inhibit electron flow through the electron transport system. As a secondary measurement, extracellular acidification rate (ECAR) was also recorded, and 2-deoxyglucose was injected at final concentration of 250 μM (port D) to interrogate the contribution of glycolysis to ECAR.

Morrissey S.M., Zhang F., Ding C., Montoya-Durango D.E., Hu X., Yang C., Wang Z., Yuan F., Fox M., Zhang H.G., Guo H., Tieri D., Kong M., Watson C.T., Mitchell R.A., Zhang X., McMasters K.M., Huang J, & Yan J. (2021). Tumor-derived Exosomes Drive Immunosuppressive Macrophages in a Pre-metastatic Niche through Glycolytic Dominant Metabolic Reprogramming. Cell metabolism, 33(10), 2040-2058.e10.

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Glycolytic Flux Profiling of Macrophages

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An XF96e Analyzer (Agilent Technologies, Santa Clara, CA) was used to measure glycolytic flux in isolated peritoneal macrophages (0.6×10⁵ cells/well). Cells were stimulated with MLE-12 or LLC exosomes (40 μg/mL) overnight with or without the presence of SEITU (2mM). The culture media was changed 1 h prior to the assay run to unbuffered Dulbecco’s Modified Eagle Medium (DMEM, pH 7.4) supplemented with 2 mM L-glutamine (Agilent cat# 103575-100). A Seahorse XF Glycolysis Stress Test Kit was used and the final concentrations of Glucose (port A), Oligomycin (port B), and 2-deoxyglucose (port C) were 10mM, 2 μM and 50mM, respectively. Three basal ECAR measurements were recorded followed by an injection of a saturating level of glucose, measuring a glucose-induced response. This was followed by Oligomycin A, an ATP synthase inhibitor, which inhibits mitochondrial ATP production and shifts the energy production to glycolysis, revealing the cellular maximum glycolytic capacity. Finally, 2-deoxyglucose, a glucose analog, was injected to inhibit glucose binding to hexokinase, thus confirming extracellular rates to be a product of glycolysis. From these experimental measurements, measures of glycolysis, glycolytic reserve, glycolytic capacity, and non-glycolytic acidification were calculated.

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Metabolic profiling of NLRP3 mutant macrophages

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Nlrp3^–/– immortalized macrophages expressing or not NLRP3 p.D303N variant and NLRP3 wild type were seeded at 25.000 cells/well in Seahorse adherent 96-well plate (Agilent Technologies) in DMEM high glucose medium (Biowest) without FBS. Cells were treated with doxycycline (1 µg/ml), LPS (100 ng/ml), MCC950 (10 µM), IL-1Ra (100 ng/ml) or 2-DG (0.1, 0.5, 1 mM) for 4 h or 16 h (as indicated in the figure legend) at 37 °C and 5% CO₂. After stimulation, cell media was discarded, and the cells were incubated with 180 µl DMEM seahorse medium supplemented with 5 mM glucose, 1 mM pyruvate and 2 mM glutamine (Agilent Technologies). The plate was incubated for 45 min at 37 °C without CO₂. Glycolytic rate assay, ATP real-time rate assay, and Cell Mito-stress kits (Agilent Technologies) were used for experiments according to the manufacturer’s instructions using an XF96e Analyzer (Agilent Technologies). After reading, the nuclei of the cells were stained with 3 mM Hoechst solution (Sigma-Aldrich) to normalize the number of cells in the calculated OCR and ECAR values. Results were collected with the Wave software version 2.6 (Agilent Technologies).

Molina-López C., Hurtado-Navarro L., García C.J., Angosto-Bazarra D., Vallejo F., Tapia-Abellán A., Marques-Soares J.R., Vargas C., Bujan-Rivas S., Tomás-Barberán F.A., Arostegui J.I, & Pelegrin P. (2024). Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production. Nature Communications, 15, 1096.

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