FAO assay using [3H]-palmitic acid was performed73 (link) with hiPSC-CMs at approximately D20 plated in a Matrigel-coated 24-well plate. Adenoviral ERRγ or GFP OE was performed as described in the hiPSC culture system section. Following ERRγ or GFP OE, cells were rinsed three times with PBS and then incubated in 125 μM [3H]-palmitic acid (PerkinElmer, NET043001MC, 60 Ci/mmol) bound to fatty acid-free albumin containing 1 mM carnitine for 2 h at 37 °C. The cell medium was transferred to a tube containing cold 10% trichloroacetic acid. The tubes were centrifuged at 8500 × g for 10 min at 4 °C. The supernatant was immediately removed, mixed with 6 N NaOH, and applied to ion-exchange resin (DOWEX 1; Sigma-Aldrich). The eluate was collected, measured by liquid scintillation analyzer (PerkinElmer) and normalized to total protein amount. The amount of cell protein was measured by Micro BCA protein assay kit (Thermo Scientific, 23235).
Net043001mc
Manufactured by PerkinElmer
Sourced in United States
NET043001MC is a laboratory equipment product by PerkinElmer. It is designed to perform a specific core function within the laboratory setting. The description of this product is limited to its primary function without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ls6500, by Beckman Coulter (3 mentions)
Net289005mc, by PerkinElmer (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Dowex 1, by Merck Group (1 mentions)
Net317250uc, by PerkinElmer (1 mentions)
Tri carb 2910 tr, by PerkinElmer (1 mentions)
Net139001mc, by PerkinElmer (1 mentions)
Tri carb 2500, by PerkinElmer (1 mentions)
8 protocols using net043001mc
1
FAO Assay in hiPSC-Derived Cardiomyocytes
2
Determination of Cellular Fatty Acid Oxidation
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The cellular FAO was determined following the published literature89 (link) Briefly, 3 × 105 cells were seeded into each well of a 12-well plate. After 12 h incubation, [9,10-3H(N)]-palmitic acid [0.5 µCi (~9.3 pmol)] (PerkinElmer, Catalog# NET043001MC) in 15 µl carrier solution (1 mM sodium palmitate, 0.17 mM BSA, 150 mM NaCl) was added to each well and incubated for 6 h. The [9,10-3H(N)]-palmitic acid was converted to 3H2O via mitochondria FAO. To control the background, each experiment contained cell-free control. After labeling, 0.5 ml out of 1 ml culture medium was collected into a 15 ml tube with 100 µl of 1.2 N HCl, and an uncapped 0.5 ml tube containing 0.25 ml of sterile distilled water was carefully inserted into the tube and further incubated for 3 days, and the radioactivity present in the water and medium were determined by scintillation counting. The FAO rate of each sample was calculated and normalized to protein concentration.
3
Quantifying Cellular Fatty Acid Metabolism
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For measuring fatty acid oxidation, BSA conjugated 2 μCi 3H-labeled oleic acid (NET289005MC, Perkin Elmer) and palmitic acid (NET043001MC, Perkin Elmer) was added into cells kept in αMEM and incubated for 6 h in the presence of 100 μM cold oleic or 50 μM palmitic acid, respectively. Then, medium was collected and extracted with phenol/chloroform to remove un-metabolized 3H-palmitic acid or oleic acid as reported before [24] (link). The 3H activity in the aqueous phase was determined by a scintillation β-counter (LS6500, Beckman).
4
Radioactive Palmitic Acid Oxidation
5
Lipid Binding Assay for TM4SF5 Proteins
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[1,2‐3H]‐Cholesterol (NET139001MC), 6,7‐3H(N)‐estradiol (NET317250UC), and 9,10‐3H(N)‐palmitic acid (NET043001MC) was purchased from Perkin Elmer Life Sciences (Waltham, MA, USA). One million Huh7 cells were seeded and transfected with streptavidin‐tagged TM4SF5 (wild‐type [WT] or mutants) for 48 h and lysed with RI lysis buffer (20 mmol/L HEPES, pH 7.4, 2 mmol/L MgCl2, 5 units/mL benzonase, and 2 mg/mL iodoacetamide). Lysed cells were collected by centrifugation at 12,000 ×g at 4°C for 10 min. The pellets were then re‐suspended in a solubilization buffer (1% n‐dodecyl‐β‐D‐maltoside [DDM], 0.1% cholesteryl hemisuccinate [CHS], 250 mmol/L NaCl, and 20 mmol/L HEPES, pH 7.4) and incubated at 4°C for 2 h. The samples were clarified by centrifugation at 12,000 ×g at 4°C for 10 min and the soluble fraction was then incubated with 50 μL streptavidin‐agarose beads at 4°C for 1 h. The beads were then washed 3 times with wash buffer (0.1% DDM, 0.01% CHS, 100 mmol/L NaCl, and 20 mmol/L HEPES, pH 7.4). Strep‐TM4SF5 conjugated beads were then incubated with 3H‐lipid at 50 μmol/L or different concentrations for 1 h. Then, the beads were washed 3 times with wash buffer, and the remaining 3H‐lipid was counted using a liquid scintillation analyzer (Tri‐Carb 2910TR, Perkin Elmer).
6
Measuring Heart FAO via Radioactive Palmitic Acid
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Heart FAO was determined by the liberation of 3H2O from [3H] palmitic acid81 (link). The reaction mixture was Krebs–Ringer bicarbonate buffer containing 74 kBq/ml 9,10(n)-[3H] palmitic acid (1.96 TBq/mmol; NET043001MC, PerkinElmer) and 4% bovine serum albumin (FA free; Applygen), which was aerated with 5% CO2–95% O2 gas for at least 20 min prior to use. Heart slices (approximately 10 mg) were incubated with the reaction mixture for 1 h at 30 °C. After the incubation period, a 2 M KCl–HCl solution was added to the reaction mixture to stop the reaction. The resulting mixture was washed with CHCl3:methanol (2:1) to remove the lipids, including the undesired radioactive materials. The radioactivity of the aqueous phase was counted using a liquid scintillation counter (Tri-Carb 2500; Perkin-Elmer)82 (link).
7
Measuring Cellular Fatty Acid Oxidation
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The protocol for measuring fatty acid oxidation has been described in our previous studies [25 (link),26 (link)]. Briefly, BSA-conjugated 2 μCi 3H-labeled oleic acid (NET289005MC, Perkin Elmer) or palmitic acid (NET043001MC, Perkin Elmer) was added into cells kept in αMEM and incubated for 6 h in the presence of cold oleic (100 μM) or palmitic (50 μM) acid, respectively. Then, medium was collected and purified with 2 steps of methanol/chloroform ((3:2) followed by (1:1)) extraction to remove un-metabolized 3H-palmitic acid or -oleic acid, as reported before [45 (link)]. The 3H radioactivity in the aqueous phase was determined using a scintillation β-counter (LS6500, Beckman) and normalized with cell lysate protein concentration to represent fatty acid β-oxidation activity of each treatment.
8
Mitochondrial β-Oxidation Assay
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This assay measures the β-oxidative activity of mitochondria by quantitation of 3H-labeled H2O generated at the acyl-CoA dehydrogenase step [23 (link)]. First, C2C12 cells were incubated in α-MEM with 2 μCi BSA conjugated 3H-labeled palmitic acid (NET043001MC, Perkin Elmer) and 50 μM palmitic acid at 37°C for 5 h. Then, the un-metabolized 3H-palmitic acid in the conditioned medium was removed by phenol/chloroform. The 3H activity in the aqueous phase was detected by a scintillation β-counter (LS6500, Beckman).
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