A single interscapular BAT depot was harvested from K15 F/F and K15-BKO mice. A 1.2mm Uni-Core biopsy puncher (Qiagen) was used to generate 5 equally sized tissue pieces per animal. Each tissue punch was placed in one well of an XF24 islet capture microplate and covered with an islet screen (Agilent). Then, 450uL of wash media (DMEM, high glucose, pyruvate (Gibco) supplemented with 25X10−3M glucose and 25X10−3M HEPES) was added to each well. Tissues pieces were washed once with wash media and then once with assay media (Seahorse XF base medium minimal DMEM supplemented (Agilent) with 25mM glucose, 1.0mM pyruvate, 3.97mM glutamine (Sigma), pH adjusted to 7.4). The plate was then incubated at 37°C for 45 min with 450uL of assay media per well. Basal oxygen consumption rate was measured with the Seahorse XFe24 Analyzer and corresponding XFe24 sensor cartridges (Agilent). Basal oxygen consumption was assessed with 10 measurement cycles, each comprised of 3 min of mixing, 2 min of waiting, and 3 min of measuring. The average of the 5 technical replicates was taken and each measurement was averaged to determine the basal oxygen consumption rate.
Seahorse xf base medium minimal dmem
Manufactured by Agilent Technologies
Seahorse XF Base Medium Minimal DMEM is a cell culture medium specifically formulated for use with Seahorse XF Analyzers. It is a minimal DMEM-based medium designed to support the measurement of cellular metabolic activity, including oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
Automatically generated - may contain errors
Lab products found in correlation
Antimycin a, by Merck Group (2 mentions)
Xf24 islet capture microplate, by Agilent Technologies (1 mentions)
Seahorse xfe24 analyzer, by Agilent Technologies (1 mentions)
Glutamine, by Merck Group (1 mentions)
Pyruvate, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Collagenase p, by Merck Group (1 mentions)
Seahorse xf calibrant solution, by Agilent Technologies (1 mentions)
L leucine, by Merck Group (1 mentions)
Fatty acid free bovine serum albumin, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Acetylcholine, by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Lonza (1 mentions)
Corm a1, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Lonza (1 mentions)
4 protocols using seahorse xf base medium minimal dmem
1
Measuring Basal Oxygen Consumption in BAT
2
Metabolic Profiling of Spheroids
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Collagenase P, d -glucose, l -leucine, l -glutamine, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and Antimycin A were purchased from Sigma Aldrich (St. Louis, MO). Fatty acid-free bovine serum albumin (BSA) was purchased from EMD Millipore (Billerica, MA). Fetal bovine serum (FBS) was obtained from Life Technologies (Carlsbad, CA). Oligomycin A was obtained from Calbiochem (San Diego, CA). Accutase was purchased from Thermo Fisher Scientific (Roskilde, Denmark). Seahorse XF96 spheroid microplates, Seahorse XF96 FluxPaks, Seahorse XF Calibrant Solution and Seahorse XF Base Medium Minimal DMEM were acquired from Agilent Technologies (Santa Clara, CA).
3
Protocols for Gaseous Carbon Monoxide Assays
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Dulbecco’s Phosphate Buffered Saline without Ca2+ and Mg2+ (DPBS; Lonza); Seahorse XF Base Medium Minimal DMEM (Agilent Technologies); acetylcholine, CORM-A1, glucose, glutamine, phenylephrine, SNP, sodium hydroxide, sodium pyruvate (Sigma-Aldrich); collagen (CHRONO-LOG); bovine serum albumin (SERVA); dimethyl sulfoxide (DMSO; Life Technologies); gaseous carbon monoxide (Multax). Inactive CORM-A1 (iCORM-A1) was prepared according to the protocol described by Motterlini et al. [12 (link)]. For the experiments with gaseous CO, an assay buffer was saturated with CO gas by bubbling for 30 min at room temperature (as described by Rotko et al. [36 (link)]) and used immediately. As solubility of CO in water is 2.3 mL/100 mL at 20 °C [37 (link)], saturation of a water–based assay buffer with pure CO gas allows to achieve at room temperature around 1 mM solution of CO.
4
Seahorse XF96 Analysis of Fatty Acid Oxidation
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The fatty acid oxidation (FAO) was measured using a microfluorimetric Seahorse XF96 Analyzer (Agilent Technologies) according to the protocol supplied by the manufacturer with minor modifications. Cells were starved with minimal substrate neurobasal-A medium (Cat# 10888022, Thermo Fisher Scientific) for 24 hours. The minimal substrate medium included 1% B-27 Supplement (Cat# 17504044, Gibco), 1 mM glutamine, 0.5 mM carnitine, and 2.5 or 0.5 mM of glucose. The day of the assay, 45 minutes prior to the assay, starved cells were washed and incubated with Seahorse XF Base medium Minimal DMEM (Cat# 102353-100, Agilent Technologies) supplemented with 2.5 or 0.5 mM glucose and 0.5 mM carnitine in a non-CO2 37°C incubator. 15 minutes prior to the assay, 40 µM etomoxir was added to the cells to measure endogenous fatty acid uptake for FAO. Palmitate-BSA or BSA control (Seahorse XF Palmitate-BSA FAO substrate, Cat# 1102720-100, Agilent Technologies) were added to cells right before initiating the XF assay. During the assay, cells were exposed to compounds in the following order: 5 µM of oligomycin (Cat# 495455, Sigma), 10 µM of FCCP [carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone] (Cat# C2920, Sigma), and 10 µM of antimycin A (Cat# A8674, Sigma) and 5 µM of rotenone (Cat# R8875, Sigma). Wave 2.6.0 (Agilent Technologies software, USA) software was used to analyze the parameters.
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