Seahorse plate

Manufactured by Agilent Technologies

Sourced in United States

Seahorse plates are specialized multi-well plates designed for use with Agilent Technologies' Seahorse XF Analyzers. These plates are engineered to facilitate the measurement of cellular metabolic activity, including oxygen consumption rate and extracellular acidification rate, within a controlled cell culture environment.

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20 protocols using seahorse plate

Monocyte Metabolism Modulation

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Monocytes were adhered to Seahorse plates as described above. Anti-MPO, anti-PR3 or isotype antibodies were added to port A of an XFe24 FluxPak. For ECAR measurements, D-glucose was added to port B. For OCR measurements rotenone or vehicle was added to port B. The Cell plate was added to the Seahorse XFe24 analyser. Six initial basal measurements were performed followed by injection of port A. For ECAR experiments three more measurements were performed before injection of glucose followed by nine further measurements. For OCR experiments one measurement was performed before rotenone injection followed by nine further measurements.

O'Brien E.C., White C.A., Wyse J., Leacy E., Porter R.K., Little M.A, & Hickey F.B. (2020). Pro-inflammatory Stimulation of Monocytes by ANCA Is Linked to Changes in Cellular Metabolism. Frontiers in Medicine, 7, 553.

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Seahorse Assay for Glycolytic Capacity

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Cells were plated on poly-L-lysine coated 96-well Seahorse plates (Seahorse Biosciences) after lentiviral infection with shNRF2 or shGFP and equilibrated for 1 h in DMEM (Sigma D6030) containing 2 mM glutamine in the absence of serum and glucose. Basal extracellular acidification rate (ECAR) was then analyzed in the Seahorse XFe96 flux analyzer (Seahorse Biosciences), followed by ECAR measurements after sequential injections of 10 mM glucose, 2 μM oligomycin and 100 mM 2-deoxyglucose (2-DG).

Bar-Peled L., Kemper E.K., Suciu R.M., Vinogradova E.V., Backus K.M., Horning B.D., Paul T.A., Ichu T.A., Svensson R.U., Olucha J., Chang M.W., Kok B.P., Zhou Z., Ihle N., Dix M.M., Jiang P., Hayward M.M., Saez E., Shaw R.J, & Cravatt B.F. (2017). Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer. Cell, 171(3), 696-709.e23.

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Metabolic Profile of Differentiated Adipocytes

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Preadipocytes were seeded onto gelatin-coated Seahorse Plates and differentiated according to standard protocols. Cells were serum-starved for 1 hour prior to the beginning of the experiment. The extracellular acidification rate (ECAR) was monitored in a Seahorse XF24 instrument using the standard glycolysis stress test protocol of 3 minutes mix, 2 minutes wait, and 3 minutes measure. For the glycolysis stress test, cells were challenged with Glucose (25 mM), Oligomycin (2μM), and 2-deoxyglucose (100 mM), allowing 4 measurements after each injection. For the oxygen consumption ratio (OCR) measurements, cells were stimulated with Oligomycin (2μM), FCCP (2μM) and antimycin A (2μM), also allowing 4 measurements after each injection.
For the normalization of respiration to protein content, cells were lysed in RIPA buffer and protein concentration was measured using the Pierce BCA kit (Thermo Fisher).

Leiria L.O., Wang C.H., Lynes M.D., Yang K., Shamsi F., Sato M., Sugimoto S., Chen E.Y., Bussberg V., Narain N.R., Sansbury B.E., Darcy J., Huang T.L., Kodani S.D., Sakaguchi M., Rocha A.L., Schulz T.J., Bartelt A., Hotamisligil G.S., Hirshman M.F., van Leyen K., Goodyear L.J., Blüher M., Cypess A.M., Kiebish M.A., Spite M, & Tseng Y.H. (2019). 12-lipoxygenase regulates cold adaptation and glucose metabolism by producing the omega-3 lipid 12-HEPE from brown fat. Cell metabolism, 30(4), 768-783.e7.

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Measurement of Brown Adipocyte Respiration

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Brown preadipocytes were seeded onto gelatin-coated Seahorse Plates and differentiated according to standard protocols. Cells were starved for 1 h and then treated for 15 min with 1 μM 12,13-diHOME or methyl acetate as vehicle. The oxygen-consumption rates (OCR) were monitored in 200 μM palmitic acid plus 100 μM albumin in a Seahorse XF24 instrument using the standard protocol of 3-min mix, 2-min wait and 3-min measure. For the normalization of respiration to protein content, cells were lysed in RIPA buffer and protein concentration was measured using the Pierce BCA kit (Life Technologies).

Lynes M.D., Leiria L.O., Lundh M., Bartelt A., Shamsi F., Huang T.L., Takahashi H., Hirshman M.F., Schlein C., Lee A., Baer L.A., May F.J., Gao F., Narain N.R., Chen E.Y., Kiebish M.A., Cypess A.M., Blüher M., Goodyear L.J., Hotamisligil G.S., Stanford K.I, & Tseng Y.H. (2017). The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue. Nature medicine, 23(5), 631-637.

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Assessing Metabolic Profiles of Activated T cells

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Naïve CD8 T cells were enriched from spleen and superficial lymph nodes using a CD8 negative selection kit (Miltenyi) to which we added CD44-biotin to further deplete CD44^hi cells (95-99% purity). Cells were stimulated for 96hr with beads coated with anti-CD3 and anti-CD28 (Miltenyi) in RPMI media supplemented with FCS (10%), L-glut, β-mercaptoethanol, and pen/strep, and ± rapa (17ng/ml) as indicated. On the day of the assay, cells were harvested and separated from dead cells by percoll gradient centrifugation and stimulation beads, washed, counted, and plated in equal numbers on Seahorse plates (Seahorse Bioscience, Massachusettes, USA) according to manufacturers protocols. Glycolysis stress test assay was run according to manufacturers protocol using glucose (10mM), oligomycin (1.5μM), and 2-DG (100mM). Mitochondrial stress test was performed using oligomycin (1.5μM), FCCP (0.75μM), Rotenone (1μM), and Antimycin A (1μM) according to manufacturers protocol.

Goldberg E.L., Smithey M.J., Lutes L.K., Uhrlaub J.L, & Nikolich-Zugich J. (2014). Immune memory-boosting dose of rapamycin impairs macrophage vesicle acidification and curtails glycolysis in effector CD8 cells, impairing defense against acute infections. Journal of immunology (Baltimore, Md. : 1950), 193(2), 757-763.

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Measuring CD8+ T Cell Glycolysis

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Purified CD8⁺ OT-I T cells (3 × 10⁵/well) were plated in 96-well Seahorse plates (Seahorse Bioscience, North Billerica, MA) previously coated with Cell-Tak (BD Biosciences, Franklin Lakes, NJ). Extracellular acidification rates (ECAR) were measured using an XF-96 extracellular flux analyzer and a glycolysis stress test kit as per the manufacturer’s instructions (Agilent, Santa Clara, CA).

Agliano F., Karginov T.A., Ménoret A., Provatas A, & Vella A.T. (2022). Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling. iScience, 25(3), 103932.

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Metabolic Profiling of OT-I Cells

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Purified OT-I cells (2 × 10⁵/well) were plated in 96-well CELL-TAK™ (BD Biosciences) coated Seahorse plates (Seahorse Bioscience; North Billerica, MA). Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using an XF-96 Extracellular Flux Analyzer and the Cell Mito Stress Test Kit (OCR) and Glycolysis Stress Test Kit (ECAR) as per the manufacturer’s instructions (Seahorse).

Tsurutani N., Mittal P., St Rose M.C., Ngoi S.M., Svedova J., Treadway F.B., Laubenbacher R., Suárez-Ramírez J.E., Cauley L.S., Adler A.J, & Vella A.T. (2015). Costimulation endows immunotherapeutic CD8 T cells with IL-36-responsiveness during aerobic glycolysis. Journal of immunology (Baltimore, Md. : 1950), 196(1), 124-134.

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Cardiomyocyte Mitochondrial Respiration Profiling

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Cardiomyocytes were isolated from 12- to 16-week-old offspring. Isolated cardiomyocytes (25,000 per well) were seeded onto laminin-coated Seahorse Plates (Agilent) according to standard protocols. The oxygen-consumption rates (OCR; indicating mitochondrial respiration) and extra-cellular acidification rates (indicating glycolysis rate) were monitored in a Seahorse XFe24 instrument using the standard protocol of 3-minute mix, 2-minute wait, and 3-minute measure [45 (link)]. Carbonyl cyanide-p-trifluoromethoxy-phenyl-hydrazon (FCCP; 2 μmol/L) was used to determine the cells' maximal respiratory capacity by allowing the electron transport chain to function at its maximal rate (maximal respiratory capacity is derived by subtracting basal respiration from the FCCP rate). Oligomycin (a complex V inhibitor; 2 μmol/L) was used to derive ATP-linked respiration and proton leak respiration (by subtracting nonmitochondrial respiration from the basal rate). AntimycinA/Rotenone (mitochondrial inhibitors; 0.5 μmol/L) was used to determine nonmitochondrial respiration. Data from wells of the same treatment group were averaged together and analyzed directly using Waves software.

Pinckard K.M., Félix-Soriano E., Hamilton S., Terentyeva R., Baer L.A., Wright K.R., Nassal D., Esteves J.V., Abay E., Shettigar V.K., Ziolo M.T., Hund T.J., Wold L.E., Terentyev D, & Stanford K.I. (2024). Maternal exercise preserves offspring cardiovascular health via oxidative regulation of the ryanodine receptor. Molecular Metabolism, 82, 101914.

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Measuring Cellular Bioenergetics with Seahorse

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Cells were plated on 96-well Seahorse plates (Agilent, Stockport, UK) at a concentration of 5000 cells per well and treated with Leflunomide (25, 50 μM) for 16 h. Seahorse cartridges were prepared according to the manufacturer’s instructions. DMEM assay medium (Agilent) was supplemented with 1 M glucose, 100 mM pyruvate, and 200 mM glutamine and used to prepare the mitochondrial assay compounds (Table 1). Once the cartridge was loaded with the appropriate compounds, the assay cartridge and cell plates were loaded into a Seahorse Xf Pro bioanalyzer and run with standard parameters. Following the assay, cells were stained with Hoechst, and assay data were normalized to the cell counts for each well.

Curel C.J., Nobeli I, & Thornton C. (2024). Leflunomide Treatment Does Not Protect Neural Cells following Oxygen-Glucose Deprivation (OGD) In Vitro. Cells, 13(7), 631.

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Real-time ATP Rate Assay of iPSM Cells

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The iPSM cells were dissociated with accutase for 5 min at 37 °C during the most efficient day of differentiation and re-seeded into fibronectin-coated Seahorse plates (Agilent) at a density of 7.27 x 10⁵ cells per cm² in 100 μL of Seahorse XF DMEM (Agilent) supplemented with 10 mM glucose (Agilent), 1 mM pyruvate (Agilent) and 2 mM glutamine (Agilent). Cells were allowed to attach at RT for 15 min and then transferred to a 37°C incubator without CO₂ for 40 min. After that time, 400 μL of Seahorse XF DMEM medium at 37°C were added carefully to each well without disturbing the attached cells for a total of 500 μL. Cells were incubated at 37°C without CO₂ for 15 more min. The Seahorse cartridge was hydrated overnight. For the real-time ATP rate assay (Agilent), 1 μM oligomycin, 0.5 μM rotenone and 0.5 μM antimycin A were used. All samples were run in seven to ten technical replicates in a Seahorse XFe24 (Agilent). Three biological replicates were performed for each species. The Wave Desktop and online app provided by the manufacturer were used for analysis.

Lázaro J., Costanzo M., Sanaki-Matsumiya M., Girardot C., Hayashi M., Hayashi K., Diecke S., Hildebrandt T.B., Lazzari G., Wu J., Petkov S., Behr R., Trivedi V., Matsuda M, & Ebisuya M. (2023). A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals. Cell Stem Cell, 30(7), 938-949.e7.

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