Seahorse xfe96 plate

Manufactured by Agilent Technologies

Sourced in United States

The Seahorse XFe96 plates are a specialized laboratory equipment used for metabolic and bioenergetic analysis. They provide a platform for measuring various parameters related to cellular respiration and glycolysis in a high-throughput manner. The plates are designed to be used in conjunction with the Seahorse XFe96 Analyzer, a dedicated instrument that monitors the real-time changes in the extracellular environment of cells.

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Seahorse XFe96 (68 citations) Seahorse plates (20 citations) XFe96 plates (15 citations) Seahorse XFe96 cell culture plate (5 citations) Seahorse XFe96 culture plate (4 citations) Seahorse XFe96 well plate (3 citations) Seahorse XFe96 FluxPak Plates (2 citations) Seahorse XFe96/XF Pro FluxPak Mini plates (2 citations)

10 protocols using seahorse xfe96 plate

Metabolic Profiling of Activated BMDMs

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For the metabolic assays, using the XFe96 metabolic analyzer (Seahorse, Agilent Technologies, Santa Clara, CA, USA) assay, 7 × 10⁴ BMDMs were plated in Seahorse XFe96 plates (V3-PS, TC treated, Agilent Technologies) and treated with the indicated activating stimuli for 24 h. Following activation, cell media was changed to XF base medium minimal DMEM (Agilent) supplemented with 10 mM glucose, 2 mM Lglutamine, 2 mM sodium pyruvate, pH adjusted to 7.4, and incubated at 37 °C, 0% CO₂ for 1 h prior to the metabolic assay. Cells were then subjected to a “mitostess” test consisting of Oligomycin (2 μΜ), FCCP (1 μM) and Rotentone/Antimycin A (2/2 μΜ) sequential injections (3 measurement cycles each), following the initial basal measurements (media-only, 3 or 4 measurement cycles). In all cases, both the oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined. The background OCR and ECAR were obtained from wells without cells (medium only), and subtracted automatically by the XFe96 software.

Christopoulos P.F., Grigalavicius M., Corthay A., Berg K, & Theodossiou T.A. (2021). Reactive Species from Two-Signal Activated Macrophages Interfere with Their Oxygen Consumption Measurements. Antioxidants, 10(7), 1149.

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Irradiation Effects on Mouse Myoblast Cells

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A mouse myoblast cell line (C2C12 [American Type Culture Collection (ATCC) CRL‐1722], ATCC, LGC standards, UK, passage 8‐11) 14 was cultured in monolayers in Dulbecco's modified eagle medium (DMEM, Gibco, Thermo‐Fischer Scientific, Waltham, Massachusetts) supplemented with 10% v/v fetal calf serum, 1% v/v penicillin/streptomycin (P/S) and 1% v/v l‐glutamine (Sigma‐Aldrich, St Louis, Missouri). Cells were seeded into 96‐well black clear bottom plates (7000 cells/well; Sigma‐Aldrich) and Seahorse XFe96 plates (10,000 cells/well; Agilent, Santa Clara, California), incubated overnight (37°C, 5% CO₂>), irradiated as described in Section 2.2.3 and changes in mitochondrial activity were assessed 8 or 24 hours post‐irradiation.

Serrage H.J., Joanisse S., Cooper P.R., Palin W., Hadis M., Darch O., Philp A, & Milward M.R. (2019). Differential responses of myoblasts and myotubes to photobiomodulation are associated with mitochondrial number. Journal of Biophotonics, 12(6), e201800411.

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Glycolysis Stress Test Using Seahorse

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Glycolysis stress testing was performed using a Seahorse XFe96 Analyzer (Agilent) as previously described (36 ). For each experiment, 4,000 cells were plated in Seahorse XFe96 plates (Agilent) and allowed to attach overnight. For inhibitor treatment experiments, media was replaced with media containing inhibitor for 6 hours. Media was aspirated, wells washed with PBS, and then 100 μL of sterile 1X Krebs-Henseleit Buffer (KHB) was added to each well for 2 hours prior to the assay period. 10 mM glucose, 1 μM oligomycin, and 10 mM 2-deoxyglucose (2-DG) were added sequentially via the assay ports with automatic mixing and three readings taken per assay stage.

Hunt B.G., Davis J.C., Fox L.H., Vicente-Muñoz S., Lester C., Wells S.I, & Waltz S.E. (2023). RON-augmented cholesterol biosynthesis in breast cancer metastatic progression and recurrence. Oncogene, 42(21), 1716-1727.

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Metabolic Profiling of Organoids

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Extracellular acidification rate (ECAR) measurements were performed using an XFe96 analyzer (Agilent Technologies). Seahorse XFe96 plates (Agilent Technologies) were coated with a 10× dilution of BME in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich). IL-17A–treated or control organoids were collected with organoid harvest solution, washed once in DMEM basic medium, resuspended in DMEM basic medium with 2 mM glutamine, and seeded onto the coated plates at 175 μl per well. Respiratory rates were measured in response to sequential injections of glucose (10 mM), oligomycin (2 μM), and 2-DG (50 mM) (all Cayman Chemical). Values were normalized to a total number of cells in organoid culture well using the CyQUANT Cell Proliferation Assay (Thermo Fisher).

Konieczny P., Xing Y., Sidhu I., Subudhi I., Mansfield K.P., Hsieh B., Biancur D.E., Larsen S.B., Cammer M., Li D., Landén N.X., Loomis C., Heguy A., Tikhonova A.N., Tsirigos A, & Naik S. (2022). Interleukin-17 governs hypoxic adaptation of injured epithelium. Science (New York, N.Y.), 377(6602), eabg9302.

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Glycolytic Flux Analysis of Cells

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Cells were seeded in Agilent Seahorse XFe96 plates at a density of 5 × 10⁴ cells/well and allowed to adhere for 8 h in a standard incubator. Cells were then equilibrated with XF base media at 37°C for 1 h in an incubator without CO₂ and then serum‐starved for 1 h in glucose‐free media‐containing treatments. Extracellular acidification rate (ECAR) was then measured using a glycolytic stress test kit (103020–100, Agilent Technologies). Briefly, the cells were treated with the sequential addition of glucose (10 mM), oligomycin (1.0 μM), and 2‐DG (50 nM), as described in the protocol of the XF glycolysis stress test using a Seahorse XFe96 Extracellular Flux Analyser (Agilent Technologies).

Yang Q., Wu F., Zhang Y, & Wang R. (2022). FOXM1 regulates glycolysis in nasopharyngeal carcinoma cells through PDK1. Journal of Cellular and Molecular Medicine, 26(13), 3783-3796.

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Glycolytic Stress Test Using Seahorse

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Cells were collected and plated in Agilent Seahorse XFe96 plates at a density of 5 × 10⁴ cells per well and allowed to adhere for 4 h in a standard incubator. Cells were next equilibrated with XF Base media at 37 °C for 1 h in an incubator lacking CO₂ and then serum-starved for 1 h in glucose-free media-containing treatments. Measurement of ECAR was performed by the Glycolytic Stress Test Kit (103020–100, Agilent Technologies, USA). Briefly, cells were subjected to sequential addition of glucose (10 mM), oligomycin (1.0 μM), and 2-DG (50 nM) according to the XF Glycolysis Stress Test protocol on a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies, USA). Data were analyzed using Wave software.

Su X., Yang Y., Yang Q., Pang B., Sun S., Wang Y., Qiao Q., Guo C., Liu H, & Pang Q. (2021). NOX4-derived ROS-induced overexpression of FOXM1 regulates aerobic glycolysis in glioblastoma. BMC Cancer, 21, 1181.

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Seahorse Assay for Oxidative Stress

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Fh1^fl/fl cells were plated at 3 × 10⁴ cells per well using 200 µl medium per well onto Seahorse XFe96 plates (Agilent) and incubated overnight at 37 °C and 5% CO₂. Then, the medium was replaced with 150 µl unbuffered Seahorse XF Base Medium (Agilent) supplemented with 5.56 mM glucose, 0.65 mM glutamine, 0.1 mM sodium pyruvate (equal to experimental medium used in other experiments) and 1% FBS (pH 7.4) and cells were placed in a CO₂-free incubator at 37 °C for 30 min. OCR was recorded using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent) at baseline and after injection of medium (untreated condition) or indicated concentrations of hydrogen peroxide. OCR was normalised to basal OCR of untreated cells.

van der Reest J., Lilla S., Zheng L., Zanivan S, & Gottlieb E. (2018). Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress. Nature Communications, 9, 1581.

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Metabolic Profiling of CD4+ T Cells

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Method as described in Alwarawrah et al. 2020 [31 (link)]. CD4⁺ T cells were washed with Seahorse XF RPMI 1640 media (Agilent, Santa Clara, CA) and plated at a density of 250,000 cells/well (50 μL) in a Seahorse XFe96 plate (Agilent) pre-coated with Cell-Tak (Corning, Corning, NY). After spinning down the plate at 200 rpm for 1 min, the plate was incubated for 30 min in a humidified 37°C incubator in the absence of CO₂. Seahorse XF RPMI 1640 media (130 μL) was added, and the plate was incubated for an additional 20 min. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe96 Analyzer (Agilent).

Kiernan K., Nichols A.G., Alwarawrah Y, & MacIver N.J. (2023). Effects of T cell leptin signaling on systemic glucose tolerance and T cell responses in obesity. PLOS ONE, 18(6), e0286470.

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Assessing Immune Cell Metabolism

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Activated or resting CD4⁺ or CD8⁺ T cells were washed with Seahorse XF RPMI 1640 media (Agilent, Santa Clara, CA) and plated at a density of 250,000 cells /well (50 μL) in a Seahorse XFe96 plate (Agilent) pre-coated with Cell-Tak (Corning, Corning, NY). After spinning down the plate at 200 rpm for 1 min, the plate was incubated for 30 min in a humidified 37^oC incubator in the absence of CO₂. Seahorse XF RPMI 1640 media (130 μL) was added and the plate was incubated for an additional 20 min. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe96 Analyzer (Agilent). Data were collected under basal conditions and after the addition of the following drugs: 1 μM oligomycin (Sigma-Aldrich), 0.5 μM flurorcarbonyl cynade phenylhydrazone (FCCP) (Sigma-Aldrich), and 0.75 μM rotenone (Sigma-Aldrich) with 1.5 μM antimycin A (Sigma-Aldrich).

Alwarawrah Y., Nichols A.G., Green W.D., Eisner W., Kiernan K., Warren J., Hale L.P., Beck M.A, & MacIver N.J. (2020). Targeting T cell oxidative metabolism to improve influenza survival in a mouse model of obesity. International journal of obesity (2005), 44(12), 2419-2429.

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Assessing Immune Cell Metabolism

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