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Unbuffered xf assay medium

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Unbuffered XF assay medium is a cell culture medium formulated for use in Seahorse XF Analyzers. It is designed to support cell metabolism and respiratory measurements without the interference of pH buffers.

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Lab products found in correlation

Xf96 polystyrene cell culture microplates, by Agilent Technologies (3 mentions) Xf96 extracellular flux analyzer, by Agilent Technologies (2 mentions) Bca assay, by Thermo Fisher Scientific (2 mentions) Bafilomycin a1, by Merck Group (2 mentions) Oligomycin, by Merck Group (2 mentions) Antimycin a, by Merck Group (2 mentions) Chloroquine, by Merck Group (2 mentions) Isoproterenol, by Merck Group (2 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions)

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XF assay medium (120 citations) Unbuffered assay medium (6 citations)

4 protocols using unbuffered xf assay medium

1

Measuring Neuronal Mitochondrial Respiration

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Mitochondrial activity was assessed using a Seahorse XF-96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) according to the manufacturer’s instructions and as described previously (Divakaruni et al., 2014 ; Yao et al., 2017 (link)). Briefly, neurons were grown in Seahorse 96-well plates (40,000 cells/well). On DIV 6, the media was changed to NB + B27 minus AOX. Neurons were treated with HU (5 μM) on DIV 7. After 24 hours, the neurons were treated with H2O2 (35 μM) for one hour. The medium was changed to unbuffered XF assay medium (Seahorse Biosciences) supplemented with 5 mM glucose, 1 mM pyruvate, and 2 mM glutamax (Invitrogen). The neurons were incubated in a non-CO2 incubator at 37oC for one hour followed by measurement of basal oxygen consumption rate (OCR) and OCR after the sequential addition of 2 μM oligomycin, 1 μM FCCP and 5 μM rotenone/antimycin A. Oligomycin inhibits mitochondrial ATP synthase activity and facilitated the measurement of ATP-linked respiration. FCCP uncouples the mitochondrial proton gradient and disrupts the mitochondrial membrane potential allowing measurement of maximal respiration. Rotenone inhibits mitochondrial complex I and antimycin inhibits mitochondrial complex III. The use of rotenone and antimycin together shuts down mitochondrial respiration and allows measurement of non-mitochondrial respiration.
Deering Brose R., Lehrmann E., Zhang Y., Reeves R.H., Smith K.D, & Mattson M.P. (2018). Hydroxyurea attenuates oxidative, metabolic, and excitotoxic stress in rat hippocampal neurons and improves spatial memory in a mouse model of Alzheimer’s disease. Neurobiology of aging, 72, 121-133.
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2

Extracellular Flux Analysis of Cellular Respiration

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Cells were plated in XF96 polystyrene cell culture microplates (Seahorse Bioscience) at a density of 30,000 cells per well. After an overnight incubation, cells were washed and preincubated for 30 minutes in unbuffered XF assay medium (Seahorse Bioscience) supplemented with 5.5 mM d-glucose and 1 mM sodium pyruvate at 37°C in a non-CO2 environment. Oxygen consumption rates were subsequently measured using an XF96 extracellular flux analyzer.
Deak A.T., Blass S., Khan M.J., Groschner L.N., Waldeck-Weiermair M., Hallström S., Graier W.F, & Malli R. (2014). IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake. Journal of Cell Science, 127(13), 2944-2955.
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3

Adipocyte Respiration and Lipid Metabolism

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iBACs were plated in XF96 polystyrene cell culture microplates (Seahorse Bioscience®, Agilent, Santa Clara, CA, USA) at a density of 10,000 cells per well and differentiated to mature adipocytes. Pretreatment with inhibitors was performed for 2 h with the following concentrations: 40 μM atglistatin [31 ], 100 nM bafilomycin A1 (Merck, Darmstadt, Germany), and 40 μM chloroquine (Merck, Darmstadt, Germany). All inhibitors and 10 μM isoproterenol (Merck, Darmstadt, Germany) were present during the 30 min equilibration in unbuffered XF assay medium (Seahorse Bioscience®, Agilent, Santa Clara, CA, USA) supplemented with 5.5–25 mM d-glucose, 2 mM glutamine and 1 mM sodium pyruvate at 37 °C in a non-CO2 environment and during measurement. Oxygen consumption rate was subsequently measured every 7 min using an XF96 extracellular flux analyzer (Seahorse Bioscience, Agilent, Santa Clara, CA, USA). Optimal concentrations of specific inhibitors/accelerators of the electron transport chain were determined in a prior titration experiments and used as followed: 2 μM oligomycin, 0.3 μM carbonyl-cyanide p-trifluoromethoxyphenylhydrazone, 2.5 μM antimycin A (all reagents form Merck, Darmstadt, Germany). Results were normalized to protein concentration determined by BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) and presented as pmol O2/(min × μg protein).
Huber K., Hofer D.C., Trefely S., Pelzmann H.J., Madreiter-Sokolowski C., Duta-Mare M., Schlager S., Trausinger G., Stryeck S., Graier W.F., Kolb D., Magnes C., Snyder N.W., Prokesch A., Kratky D., Madl T., Wellen K.E, & Bogner-Strauss J.G. (2018). N-acetylaspartate pathway is nutrient responsive and coordinates lipid and energy metabolism in brown adipocytes. Biochimica et biophysica acta. Molecular cell research, 1866(3), 337-348.
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4

Adipocyte Respiration and Lipid Metabolism

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iBACs were plated in XF96 polystyrene cell culture microplates (Seahorse Bioscience®, Agilent, Santa Clara, CA, USA) at a density of 10,000 cells per well and differentiated to mature adipocytes. Pretreatment with inhibitors was performed for 2 h with the following concentrations: 40 μM atglistatin [31 ], 100 nM bafilomycin A1 (Merck, Darmstadt, Germany), and 40 μM chloroquine (Merck, Darmstadt, Germany). All inhibitors and 10 μM isoproterenol (Merck, Darmstadt, Germany) were present during the 30 min equilibration in unbuffered XF assay medium (Seahorse Bioscience®, Agilent, Santa Clara, CA, USA) supplemented with 5.5–25 mM d-glucose, 2 mM glutamine and 1 mM sodium pyruvate at 37 °C in a non-CO2 environment and during measurement. Oxygen consumption rate was subsequently measured every 7 min using an XF96 extracellular flux analyzer (Seahorse Bioscience, Agilent, Santa Clara, CA, USA). Optimal concentrations of specific inhibitors/accelerators of the electron transport chain were determined in a prior titration experiments and used as followed: 2 μM oligomycin, 0.3 μM carbonyl-cyanide p-trifluoromethoxyphenylhydrazone, 2.5 μM antimycin A (all reagents form Merck, Darmstadt, Germany). Results were normalized to protein concentration determined by BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) and presented as pmol O2/(min × μg protein).
Huber K., Hofer D.C., Trefely S., Pelzmann H.J., Madreiter-Sokolowski C., Duta-Mare M., Schlager S., Trausinger G., Stryeck S., Graier W.F., Kolb D., Magnes C., Snyder N.W., Prokesch A., Kratky D., Madl T., Wellen K.E, & Bogner-Strauss J.G. (2018). N-acetylaspartate pathway is nutrient responsive and coordinates lipid and energy metabolism in brown adipocytes. Biochimica et biophysica acta. Molecular cell research, 1866(3), 337-348.
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