Respirometry was performed using a protocol adapted from (Wikstrom et al., 2012 (link)). Briefly, islets were isolated and pooled from 10 mice per group, then allowed to recover overnight as for GSIS assays. Islets were then starved in Seahorse XF DMEM Assay Medium supplemented with 1% FBS, 2.8 mM glucose, and 5 mM HEPES (final pH of 7.4) at 37°C without CO2 for 45 min. Following starvation, islets were seeded at a density of 100 size-matched islets per well into the depressions of XF24 Islet Capture Microplates containing Seahorse XF DMEM Assay Medium supplemented as above, then capture screens were inserted into wells. The plate was then loaded into the Seahorse Bioscience XF24 Extracellular Flux Analyzer heated to 37°C, and oxygen consumption was measured sequentially for the following states: basal (2.8 mM glucose), nutrient-stimulated (16.8 mM glucose or 10 mM leucine/glutamine), ATP synthase-independent (5 mM oligomycin), and non-mitochondrial (0.5 μM rotenone and 2 μM antimycin) respiration. Maximum uncoupled respiration was measured by treating with 2 μM FCCP after the addition of leucine/glutamine and oligomycin. Following respirometry, wells were imaged and relative islet area was calculated using ZEN. Oxygen consumption rates for each well were normalized to islet area relative to that of all wells.
Dmem xf assay medium
Manufactured by Agilent Technologies
Sourced in United States
DMEM XF Assay medium is a cell culture medium designed for use in Extracellular Flux (XF) analysis. It is a formulation optimized for cellular respiration measurements.
Automatically generated - may contain errors
6 protocols using dmem xf assay medium
1
Islet Respirometry Assay Adapted from Wikstrom et al.
2
Islet Respirometry Assay Adapted from Wikstrom et al.
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Respirometry was performed using a protocol adapted from (Wikstrom et al., 2012 (link)). Briefly, islets were isolated and pooled from 10 mice per group, then allowed to recover overnight as for GSIS assays. Islets were then starved in Seahorse XF DMEM Assay Medium supplemented with 1% FBS, 2.8 mM glucose, and 5 mM HEPES (final pH of 7.4) at 37°C without CO2 for 45 min. Following starvation, islets were seeded at a density of 100 size-matched islets per well into the depressions of XF24 Islet Capture Microplates containing Seahorse XF DMEM Assay Medium supplemented as above, then capture screens were inserted into wells. The plate was then loaded into the Seahorse Bioscience XF24 Extracellular Flux Analyzer heated to 37°C, and oxygen consumption was measured sequentially for the following states: basal (2.8 mM glucose), nutrient-stimulated (16.8 mM glucose or 10 mM leucine/glutamine), ATP synthase-independent (5 mM oligomycin), and non-mitochondrial (0.5 μM rotenone and 2 μM antimycin) respiration. Maximum uncoupled respiration was measured by treating with 2 μM FCCP after the addition of leucine/glutamine and oligomycin. Following respirometry, wells were imaged and relative islet area was calculated using ZEN. Oxygen consumption rates for each well were normalized to islet area relative to that of all wells.
3
Measuring Adipocyte Bioenergetics in vitro
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Primary brown adipocytes were cultured and differentiated on a XF96e-well plate. At day 5 of differentiation, cells were washed and incubated in DMEM XF Assay medium (Seahorse Bioscience, Santa Clara, CA, USA), supplemented with 25 mM glucose (Carl Roth, Karlsruhe, Germany) and 1.5% fatty acid free-BSA (Sigma-Aldrich, Munich, Germany) at 37 °C in a non-CO2 incubator for 10 min. Tenfold higher concentrated compounds, dissolved in DMEM XF Assay medium without supplements, were loaded into the ports of a XF Assay Cartridge. Oxygen consumption rate (OCR) was measured using an extracellular flux analyzer (XF96, Seahorse Bioscience, Santa Clara, CA, USA). Basal OCR was recorded for 9 min, followed by measurement of OCR after injection of oligomycin (2 µg/ml, 23 min), norepinephrine (1 µM, 27 min), icilin (1 µM, 27 min) or control (0.1% DMSO, 27 min), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1 µM, 14 min), rotenone (2.5 µM)/ antimycin A (2.5 µM)/2-deoxyglucose (10 mM) (9 min). For normalization, the cell plate was fixed with 4% paraformaldehyde and subsequently co-stained with 4′,6-diamidino-2-phenylindole (Dapi) and Nile red. Using a PheraStar plate reader, the fluorescence signal was detected and the bioenergetics measurements were corrected for cell number and differentiation.
4
Metabolic Profiling of Primary Adipocytes
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Primary iWAT cells were isolated, macrophage-depleted and differentiated
for 5 d on a collagen-coated XF96 well plate. On the day of the experiment (Day
5), cells were washed with DMEM XF Assay medium (Seahorse Bioscience, Santa
Clara, CA), supplemented with 25 mM glucose, 10 mM pyruvate and 0.3% fatty-acid
free BSA, and incubated in 180 µl of XF Assay medium in a non-CO2
incubator at 37°C for 1 h. All port compounds were dissolved in pure DMEM
XF Assay medium without supplements and 10-fold higher concentrated compounds
were loaded into the ports of a XF Assay Cartridge. Oxygen consumption rate
(OCR) was measured using an extracellular flux analyzer (XF96, Seahorse
Bioscience, Santa Clara, CA). Basal OCR was recorded for 21 min followed by
measurement of OCR after injection of isoproterenol (1 µM, 35 min),
oligomycin (2 µg/ml, 21 min), carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1 µM, 21 min), rotenone
(2.5 µM)/antimycin (2.5 µM)/2-Deoxyglucose (10 mM) (28 min). For
normalization, the cell plate was subsequently co-stained with Dapi and Nile red
and the fluorescence signal was detected to correct for cell number and
differentiation.
for 5 d on a collagen-coated XF96 well plate. On the day of the experiment (Day
5), cells were washed with DMEM XF Assay medium (Seahorse Bioscience, Santa
Clara, CA), supplemented with 25 mM glucose, 10 mM pyruvate and 0.3% fatty-acid
free BSA, and incubated in 180 µl of XF Assay medium in a non-CO2
incubator at 37°C for 1 h. All port compounds were dissolved in pure DMEM
XF Assay medium without supplements and 10-fold higher concentrated compounds
were loaded into the ports of a XF Assay Cartridge. Oxygen consumption rate
(OCR) was measured using an extracellular flux analyzer (XF96, Seahorse
Bioscience, Santa Clara, CA). Basal OCR was recorded for 21 min followed by
measurement of OCR after injection of isoproterenol (1 µM, 35 min),
oligomycin (2 µg/ml, 21 min), carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1 µM, 21 min), rotenone
(2.5 µM)/antimycin (2.5 µM)/2-Deoxyglucose (10 mM) (28 min). For
normalization, the cell plate was subsequently co-stained with Dapi and Nile red
and the fluorescence signal was detected to correct for cell number and
differentiation.
5
Metabolic Profiling of Primary Adipocytes
6
Adipocyte Respiration Profiling
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Primary BAT cells were isolated and differentiated for 5 d on a collagen-coated XF24 well plate. On Day 5, cells were washed with DMEM XF Assay medium (Seahorse Bioscience, Santa Clara, CA, USA), supplemented with 25 mM glucose, no pyruvate and no fatty-acid free BSA. All port compounds were dissolved in pure DMEM XF Assay medium without supplements and 10-fold higher concentrated compounds were loaded into the ports of a XF Assay Cartridge. Oxygen consumption rate (OCR) was measured using an extracellular flux analyzer (XF24, Seahorse Bioscience, Santa Clara, CA, USA). Basal OCR was recorded for 15 min followed by measurement of OCR after injection of oligomycin (5 µg ml−1, 15 min), isoproterenol (1 µM, 15 min), 2,4-Dinitrophenol (DNP) (100 µM, 9 min), rotenone (5 µM)/antimycin (2 µM)/2-Deoxyglucose (100 mM) (15 min). For normalization, the cell plate was subsequently co-stained with Dapi and Nile red and the fluorescence signal was detected to correct for cell number and differentiation. Proton leak respiration was calculated by subtraction of Rot/AA/2DG-OCR from Oligomycin-OCR.
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