Seahorse xfp extracellular flux analyzer

Manufactured by Agilent Technologies

Sourced in United States, United Kingdom

The Seahorse XFp Extracellular Flux Analyzer is a compact, bench-top instrument designed to measure the metabolic activity of cells. It quantifies the oxygen consumption rate and extracellular acidification rate of live cells in a microplate format.

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107 protocols using seahorse xfp extracellular flux analyzer

Mitochondrial Function Profiling via Seahorse Analyzer

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Oxygen consumption rate (OCR, indicative of mitochondrial OXPHOS) in day one sterilized animals was measured using the Seahorse XFp Extracellular Flux Analyzer (Agilent Seahorse Technologies) adapting the protocol used to measure OCR in C. elegans using a Seahorse XF96 Extracellular Flux Analyzer (Koopman et al., 2016 (link)). Each assay run compared the OCR between two genotypes, in three wells each and five measurements were made for each well for basal OCR and maximal OCR upon addition of FCCP. Assays were repeated on three separate days and three biological replicates were analyzed.
Mitochondrial OXPHOS in human fibroblasts (AG08379 and AG06848) was analyzed using a Seahorse XFp Extracellular Flux Analyzer (Agilent Seahorse Technologies) by measuring the OCR in real time. For OCR analysis, cells were seeded in 8-well plates designed for XFp at 25,000 cells per well in complete growth media. On the next day, the cells were switched to unbuffered media (supplemented with 2.5 mM glucose, 1 mM pyruvate, and 1 mM glutamine) and further incubated in a CO2 -free incubator for 1 hr prior to measurement. During measurement, oligomycin (1 µM), FCCP (1 µM), antimycin A and rotenone (0.5 µM) were added. At the end of each assay, protein quantification was performed for normalization. All experiments were performed in triplicate and repeated three times.

Sarasija S., Laboy J.T., Ashkavand Z., Bonner J., Tang Y, & Norman K.R. (2018). Presenilin mutations deregulate mitochondrial Ca2+ homeostasis and metabolic activity causing neurodegeneration in Caenorhabditis elegans. eLife, 7, e33052.

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Measuring Cellular Respiration using Seahorse

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The oxygen consumption rate (OCR) of whole cells was determined by using a Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience). Cells were trypsinized and plated on a miniplate 24 hours prior to the Seahorse assay. For 2DG experiments, the OCR was normalized to the final cell number determined by manual cell counting. For DNP experiments, the OCR was monitored upon injections of 20 μM DNP or 100 μM DNP.

Lee J., Prohaska J.R, & Thiele D.J. (2001). Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development. Proceedings of the National Academy of Sciences of the United States of America, 98(12), 6842-6847.

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Evaluating Mitochondrial Function in Cells

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Oxygen consumption rates were measured using the Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s instructions. For OCR experiments, cells were cultured in 4 mM glutamine, 5% FBS and the indicated glucose levels for 24–36 h. Mitochondrial membrane potential was measured with tetramethyl rhodamine ethyl ester (TMRE) staining (Abcam, no. ab113852) according to the manufacturer’s instructions. Mitochondrial mass was measured with a mitochondrial marker, mitotracker green (Thermo Fisher Scientific, no. M7514), using two different approaches: a plate reader and live confocal imaging. NADPH (Abcam, no. ab65349), GSH/GSSG ratio (Promega, no. V6611) and ATP (Abcam, no. 83355) measurements were performed according to the manufacturer’s instructions. All readouts were normalized to cell number. For AG-120 experiments, cells were treated with AG-120 (2 µM) under 4 mM glutamine, 5% FBS and indicated glucose levels.

Vaziri-Gohar A., Cassel J., Mohammed F.S., Zarei M., Hue J.J., Hajihassani O., Graor H.J., Srikanth Y.V., Karim S.A., Abbas A., Prendergast E., Chen V., Katayama E.S., Dukleska K., Khokhar I., Andren A., Zhang L., Wu C., Erokwu B., Flask C.A., Zarei M., Wang R., Rothermel L.D., Romani A.M., Bowers J., Getts R., Tatsuoka C., Morton J.P., Bederman I., Brunengraber H., Lyssiotis C.A., Salvino J.M., Brody J.R, & Winter J.M. (2022). Limited nutrient availability in the tumor microenvironment renders pancreatic tumors sensitive to allosteric IDH1 inhibitors. Nature Cancer, 3(7), 852-865.

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Seahorse XFp Metabolic Analysis

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Metabolic measurements were performed using the Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience; North Billerica, Billerica, MA, USA). For this, myoblasts were seeded in XFp Cell Culture Miniplates (103025-100, Seahorse Bioscience), at a density of 1.5 × 10⁴ cells per well and incubated overnight.
To investigate glycolytic function cells were incubated in unbuffered Basal Assay Medium (Seahorse) supplemented with 1 mM glutamine pH 7.4 at 37 °C without CO₂ for 1 h before the assay. Following sequential injection of glucose (10 mM), oligomycin (1.0 μM), and 2-deoxy-d-glucose (50 mM) extracellular acidification rate (ECAR) was measured. Analysis of the data was performed well-wise.

Hintze S., Limmer S., Dabrowska-Schlepp P., Berg B., Krieghoff N., Busch A., Schaaf A., Meinke P, & Schoser B. (2020). Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease. International Journal of Molecular Sciences, 21(7), 2642.

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Measuring Mitochondrial Respiration in OVCAR-8 Cells

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OVCAR-8 cells were plated at 8,000 cells/well onto Seahorse 8-well XFp cell culture miniplates (Cat. No. 103025-100, Seahorse Bioscience, Agilent Technologies, Santa Clara, CA) and allowed to grow for 24 hours before being assayed for oxygen consumption rate (OCR) on a Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience, Agilent Technologies, Santa Clara, CA). One hour prior to the start of the assay, cells were washed and changed to Seahorse XF base assay medium supplemented with 2 mmol/L l-glutamine and 10 mmol/L glucose, adjusted to pH 7.4, and incubated in a 37°C non-CO₂ incubator. OCR was measured over 6 hours under basal conditions and after the addition of VLX600 (40 nM or 5 uM).

Ekstrom T.L., Pathoulas N.M., Huehls A.M., Kanakkanthara A, & Karnitz L.M. (2021). VLX600 Disrupts Homologous Recombination and Synergizes with PARP Inhibitors and Cisplatin by Inhibiting Histone Lysine Demethylases. Molecular Cancer Therapeutics, 20(9), 1561-1571.

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Evaluating Mitochondrial Function in Hypoxia

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The mitochondrial OCR and ECAR were measured using Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) as described previously^{33 (link)}. Briefly, 3 × 104 cells were seeded in each well of Seahorse XFp Cell Culture Miniplates coated with poly-L-lysine solution (Sigma-Aldrich, St. Louis, MO) two days prior to the assay. The cells were exposed to cobalt chloride (CoCl2) (Sigma-Aldrich) at 100 μM final concentration for 16hr to create the mimic hypoxia. Subsequently, the cells were treated with or without YC-1 (10 μM) for 24 h. Before performing the glycolysis stress test, the culture medium was removed from each well, and then the cells were washed 2 times and filled by the assay medium for Seahorse adjusted the pH to 7.4 ( ± 0.02). Thereafter, the glycolysis stress test was employed by sequential injections of glucose (10 mM), oligomycin (2.5 μM) and 2-deoxy-glucose (2-DG) (50 mM). OCR and ECAR under basal and glucose-stimulated conditions were evaluated as means of values at the three time points before and after the addition of glucose, respectively.

Wakiyama K., Kitajima Y., Tanaka T., Kaneki M., Yanagihara K., Aishima S., Nakamura J, & Noshiro H. (2017). Low-dose YC-1 combined with glucose and insulin selectively induces apoptosis in hypoxic gastric carcinoma cells by inhibiting anaerobic glycolysis. Scientific Reports, 7, 12653.

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Seahorse Extracellular Flux Analysis of Mitochondrial Function

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OCRs were measured in mPHs according to the manufacturer’s recommended protocol of Seahorse XFp Extracellular Flux Analyzer (SeahorseBioscience, MA). The cells were harvested into collagen-coated plates (2 × 10⁴ cells/well) in DMEM containing 10% FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin. One hour before the experiment, cells were washed with an XF assay base medium and placed into a non-CO₂ incubator maintained at 37 °C before completion of probe cartridge calibration. During the time course of the experiment, the oxygen concentration was measured overtime periods of 3 min at 3-min intervals. The rates of basal, uncoupled (by addition of 2 μM oligomycin), maximal (with 2 μM FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone), and non-mitochondrial respiration (with rotenone plus antimycin A, 0.5 μM each) were measured using an XFp Extracellular Flux Analyzer.

Kim Y.S., Ko B., Kim D.J., Tak J., Han C.Y., Cho J.Y., Kim W, & Kim S.G. (2022). Induction of the hepatic aryl hydrocarbon receptor by alcohol dysregulates autophagy and phospholipid metabolism via PPP2R2D. Nature Communications, 13, 6080.

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Measuring Cellular Oxygen Consumption Rate

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The oxygen consumption rate (OCR) of whole cells was determined by using the Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience). Cells were trypsinized and plated on a miniplate 24 hr prior to the Seahorse assay. For Mfn2 knockdowns, cells were treated with scrambled siRNA as a control or Mfn2 siRNA for 48 hr prior to seeding. The assay medium consisted of 25 mM glucose, 4 mM glutamine, 50 μM palmitate-BSA, and 50 μM oleate-BSA in Seahorse base medium. The OCR was monitored upon serial injections of oligomycin (oligo, 2 μM), FCCP (1 μM), and a rotenone/antimycin A mixture (rot/AA, 1 μM). A concentration of 1 μM FCCP was determined to be optimal in separate experiments. OCR was normalized to the final cell number or total protein amount as determined by manual cell counting or by using a BCA assay, respectively. Data presented have been corrected for non-mitochondrial respiration.

Yao C.H., Wang R., Wang Y., Kung C.P., Weber J.D, & Patti G.J. (2019). Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation. eLife, 8, e41351.

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Metabolic Profiling of TGF-β Treatment

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Cells were cultured in eight‐well XF assay plates and then treated with or without TGF‐β (5 ng·mL⁻¹) in the presence or absence of FF (25 μm) or WY14643 (50 μm) for 48 h. The oxygen concentration rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA).

Kikuchi R., Maeda Y., Tsuji T., Yamaguchi K., Abe S., Nakamura H, & Aoshiba K. (2021). Fenofibrate inhibits TGF‐β‐induced myofibroblast differentiation and activation in human lung fibroblasts in vitro. FEBS Open Bio, 11(8), 2340-2349.

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Nebivolol Modulates Cellular Respiration

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The OSCC cells were reseeded in 24-well microplates which were treated with and without 10 µM nebivolol for 2 h. The corresponding treated cells’ oxygen consumption rate (OCR) was measured by Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience). The sequential final concentrations of injected substances were 2.5 μM oligomycin, 1 μM FCCP, 1 μM rotenone, and antimycin. Each group was determined five times and normalized according to the number of cells.

Chen Q., Jiang H., Wang Z., Cai L.Y., Jiang Y.C., Xie L., Zhou Y., Zeng X., Ji N., Shen Y.Q, & Chen Q.M. (2021). Adrenergic Blockade by Nebivolol to Suppress Oral Squamous Cell Carcinoma Growth via Endoplasmic Reticulum Stress and Mitochondria Dysfunction. Frontiers in Pharmacology, 12, 691998.

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