Seahorse xf dmem media

Manufactured by Agilent Technologies

Seahorse XF DMEM media is a specialized cell culture medium designed for use with Agilent's Seahorse XF Analyzers. It is formulated to support the metabolic analysis of cells during Seahorse XF experiments.

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6 protocols using seahorse xf dmem media

Mitochondrial Respiration Analysis in Lymphoma

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OCI-Ly and SU-DHL4 cells were counted and plated on a Seahorse 96-well plate at a density of 1.25×10⁵ or 5×10⁵ cells, respectively in Seahorse XF DMEM media (Agilent). OCR and ECAR were simultaneously recorded using a Seahorse XF96 Analyzer (Agilent) for 12-38 consecutive measurements. OCR is measured before and after the addition of inhibitors to assess mitochondrial function by deriving several parameters of mitochondrial respiration: (i) basal respiration, (ii) ATP-linked respiration and proton leak respiration (after 3 μM oligomycin [Sigma], a complex V inhibitor) and (iii) maximal respiration (after 1 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) [Sigma], a protonophore). Mitochondrial respiration is finally inhibited by 1 μM antimycin A (Sigma), a complex III inhibitor.
To measure the effect of venetoclax on OCR/ECAR, cells were pre-treated for 1h with 10μM of zVAD-FMK (Abcam) or left untreated and OCR/ECAR were recorded for 4 measurements before injection of venetoclax at a final concentration of 100 nM. DMSO injection was used as control.

Guièze R., Liu V.M., Rosebrock D., Jourdain A.A., Hernández-Sánchez M., Zurita A.M., Sun J., Hacken E.T., Baranowski K., Thompson P.A., Heo J.M., Cartun Z., Aygün O., Iorgulescu J.B., Zhang W., Notarangelo G., Livitz D., Li S., Davids M.S., Biran A., Fernandes S.M., Brown J.R., Lako A., Ciantra Z.B., Lawlor M.A., Keskin D.B., Udeshi N.D., Wierda W.G., Livak K.J., Letai A.G., Neuberg D., Wade Harper J., Carr S.A., Piccioni F., Ott C.J., Leshchiner I., Johannessen C.M., Doench J., Mootha V.K., Getz G, & Wu C.J. (2019). Mitochondrial reprogramming underlies resistance to BCL-2 inhibition in lymphoid Malignancies. Cancer cell, 36(4), 369-384.e13.

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Metabolic Profiling of Cell Lines

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Measurements of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were obtained utilizing the Seahorse XFe96 Analyzer (Seahorse Biosciences) as previously described (Pike Winer and Wu, 2014 ). In brief, cells were seeded in their respective, fully supplemented medium at a range of densities optimized for each PDCL. 45 minutes prior to starting the assay, cells were equilibrated in seahorse XF DMEM media (Agilent, cat# 103575–100) supplemented with 2mM L-glutamine at 37°C in a non-CO2 incubator. During the assay, indicated compounds were injected into wells at 18-minute intervals. All results were normalized to total cellular protein content per well by RIPA extraction followed quantification with BCA protein assay kit (ThermoFisher Scientific, #23227,) in a 96-well format, with absorbance measured using a Tecan Infinite 200 plate-reader.

Brunton H., Caligiuri G., Cunningham R., Upstill-Goddard R., Bailey U.M., Garner I.M., Nourse C., Dreyer S., Jones M., Moran-Jones K., Wright D.W., Paulus-Hock V., Nixon C., Thomson G., Jamieson N.B., McGregor G.A., Evers L., McKay C.J., Gulati A., Brough R., Bajrami I., Pettitt S.J., Dziubinski M.L., Barry S.T., Grützmann R., Brown R., Curry E., Pajic M., Musgrove E.A., Petersen G.M., Shanks E., Ashworth A., Crawford H.C., Simeone D.M., Froeling F.E., Lord C.J., Mukhopadhyay D., Pilarsky C., Grimmond S.E., Morton J.P., Sansom O.J., Chang D.K., Bailey P.J, & Biankin A.V. (2020). HNF4A and GATA6 Loss Reveals Therapeutically Actionable Subtypes in Pancreatic Cancer. Cell reports, 31(6), 107625.

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Mitochondrial Respiration Profiling

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1.25 × 10⁵ K562 cells were plated on a Seahorse plate in Seahorse XF DMEM media (Agilent) containing 25mM glucose and 4mM glutamine (ThermoFisher Scientific). Oxygen consumption and extracellular acidification rates were simultaneously recorded by a Seahorse XFe96 Analyzer (Agilent) using the mito stress test protocol, in which cells were sequentially perturbed by 2mM oligomycin, 1μM CCCP and 0.5mM antimycin (Sigma). Data were analyzed using the Seahorse Wave Desktop Software (Agilent). Data were not corrected for carbonic acid derived from respiratory CO₂. For seahorse in HeLa and HAP1 cells, 5 × 10⁴ and 1 × 10⁵ cells, respectively, were plated in a 96-well seahorse plate the day before the experiment. Cells were trypsinized after the experiment and recounted and the data was normalized to cell number.

Jourdain A.A., Begg B.E., Mick E., Shah H., Calvo S.E., Skinner O.S., Sharma R., Blue S.M., Yeo G.W., Burge C.B, & Mootha V.K. (2021). Loss of LUC7L2 and U1 snRNP Subunits Shifts Energy Metabolism from Glycolysis to OXPHOS. Molecular cell, 81(9), 1905-1919.e12.

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Metabolic Profiling of Cell Lines

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Respiratory Profile of Pancreatic Cell Lines

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Sphere-derived Panc185, PancA6L and Panc215 cells were plated in XF HS Miniplates (Seahorse Bioscience) at a cellular density of 5,000 cells/well. For OCR determination, cells were incubated in Seahorse XF DMEM media (103680, Agilent) supplemented with 2mM glutamine, 10mM glucose, and 1mM pyruvate for 1 h, prior to the measurements using the Seahorse XFp Cell Mito Stress Kit (103010, Agilent). After an OCR baseline measurement, the minimum oxygen consumption was determined adding 1.5µM oligomycin (O) and the maximal respiration rate was assessed by adding 1µM FCCP (F). At the end of the experiment the non-mitochondrial oxygen consumption was evaluated adding both 0.5µM rotenone (R) and antimycin (A). Experiments were run in a XF HS Mini analyzer (Seahorse Agilent), and raw data were normalized to total protein using BCA protein assay kit (Cat. no. 23225, Thermo Scientific).

Alcalá S., Villarino L., Ruiz-Cañas L., Couceiro J.R., Martínez-Calvo M., Palencia-Campos A., Navarro D., Cabezas-Sainz P., Rodriguez-Arabaolaza I., Cordero-Barreal A., Trilla-Fuertes L., Rubiolo J.A., Batres-Ramos S., Vallespinos M., González-Páramos C., Rodríguez J., Gámez-Pozo A., Vara J.Á., Fernández S.F., Berlinches A.B., Moreno-Mata N., Redondo A.M., Carrato A., Hermann P.C., Sánchez L., Torrente S., Fernández-Moreno M.Á., Mascareñas J.L, & Sainz B J.r. (2024). Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy. Journal of Experimental & Clinical Cancer Research : CR, 43, 33.

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Metabolic Profiling of Muropeptide Effects

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OCR measurements were performed with a Seahorse XF-24 analyzer (Seahorse Bioscience). Cells were seeded in a Seahorse XF-24 cell culture microplate [1.7 ×10⁵ caco-2 cells per well in 250 μL DMEM media (Gibco, 11995040) supplemented with 10% heat-in-activated FBS and 1% non-essential amino acid, or 1.0 ×10⁴ fibroblast cells per well in 200 μL EMEM media supplemented with 15% FBS] and then incubated at 37°C and 5% CO₂ overnight. After overnight recovery, the cells were incubated with muropeptide solution or lysozyme solution (mock) for 24 h. Cells were washed in Seahorse XF DMEM media (Agilent, 30119005, supplemented with 1mM pyruvate, 10 mM glucose and 2 mM glutamine) and incubated at 37°C with no CO₂ for 1 h before measurement. Cells were washed again with Seahorse XF DMEM media just before measuring. OCR was measured and analyzed by adding 2.5 μM oligomycin, 1μM FCCP and 1.25 μM ROT/AA (Agilent, 103015–100) sequentially following the manufacture’s instruction. After OCR measurements, cells from each well were lysed in lysis buffer (100 mM NaCl, 0.5mM EDTA, 0.5% NP-40 and a cocktail of protease inhibitors in 10 mM Tris-HCl, pH7.4) and total proteins were quantified by using the BCA analysis.

Tian D., Cui M, & Han M. (2024). Bacterial muropeptides promote OXPHOS and suppress mitochondrial stress in mammals. Cell reports, 43(4), 114067.

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