FAO assay was performed as described previously (Ye et al., 2016 (link)). Briefly, sorted Lin− WT and NOX2 KO leukemia cells were cultured in IMDM containing 10 μM palmitate (Sigma) and 10% FBS (Thermo Scientific) overnight and then equal numbers of cells were plated in 48-well plates supplemented with FAO assay medium (IMDM containing 2.5% FBS, 10 μM palmitic acid, 1% fatty acid free BSA (Sigma), 500 μM carnitine (Sigma). Cells were pulsed for 4 hours with 0.5 μCi [9,10-3H(N)]-palmitic acid (Perkinelmer) and the medium was collected to analyze the released 3H2O, formed during cellular oxidation of [3H] palmitate. Briefly, medium was precipitated by 10% trichloroacetic acid (Sigma) and then supernatant was neutralized with 6N NaOH (Sigma) and loaded into ion exchange columns packed with DOWEX 1X2-400 resin (Sigma). The radioactive product was eluted with water and quantitated by liquid scintillation counting.
Dowex 1x2 400 resin
Manufactured by Merck Group
DOWEX 1X2-400 is a strong anion exchange resin. It is a macroporous, gel-type resin that is used for the removal of ionic contaminants from aqueous solutions. The resin is made up of a polystyrene-divinylbenzene matrix with quaternary ammonium functional groups.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Carnitine, by Merck Group (3 mentions)
9 10 3h n palmitic acid, by PerkinElmer (3 mentions)
6n naoh, by Merck Group (3 mentions)
Fatty acid free bsa, by Merck Group (3 mentions)
Palmitic acid, by Merck Group (3 mentions)
Trichloroacetic acid, by Merck Group (3 mentions)
Palmitate, by Merck Group (2 mentions)
1 14c acetic acid, by PerkinElmer (1 mentions)
4 protocols using dowex 1x2 400 resin
1
FAO Assay for Leukemia Cell Metabolism
2
Fatty Acid Oxidation Measurement in CD8+ T Cells
3
Assays for Cellular Fatty Acid Oxidation and Lipogenesis
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For fatty acid oxidation, primary MEFs were serum starved for 3 h and incubated overnight in culture medium containing 100 mM palmitate (C16:0) and 1 mM carnitine. In the final 2 h of incubation, cells were pulsed with 1.7 µM Ci[9,10(n)-3H] palmitic acid (GE Healthcare), and the medium was collected and eluted on ion exchange columns packed with DOWEX 1X2-400 resin (Sigma) to analyze the released 3H2O, formed during cellular oxidation of [3H] palmitate. Primary MEFs used for this assay were below passage 5.
For the measurement of lipogenesis, A549 cells were cultured, placed overnight in low-glucose low-serum medium, then labeled with 1-14C acetic acid (Perkin Elmer) while stimulated with serum-containing medium for 1 h. Cells were washed twice with PBS before lysis in 0.5% Triton X-100. The lipid fraction was isolated by the addition of chloroform and methanol (2:1 [v/v]), followed by the addition of water. Samples were centrifuged, and 14C incorporation was measured in the bottom, lipid-containing phase using a scintillation counter. Each condition was normalized to protein concentrations.
For the measurement of lipogenesis, A549 cells were cultured, placed overnight in low-glucose low-serum medium, then labeled with 1-14C acetic acid (Perkin Elmer) while stimulated with serum-containing medium for 1 h. Cells were washed twice with PBS before lysis in 0.5% Triton X-100. The lipid fraction was isolated by the addition of chloroform and methanol (2:1 [v/v]), followed by the addition of water. Samples were centrifuged, and 14C incorporation was measured in the bottom, lipid-containing phase using a scintillation counter. Each condition was normalized to protein concentrations.
4
FAO Assay for Leukemia Cell Metabolism
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FAO assay was performed as described previously (Ye et al., 2016 (link)). Briefly, sorted Lin− WT and NOX2 KO leukemia cells were cultured in IMDM containing 10 μM palmitate (Sigma) and 10% FBS (Thermo Scientific) overnight and then equal numbers of cells were plated in 48-well plates supplemented with FAO assay medium (IMDM containing 2.5% FBS, 10 μM palmitic acid, 1% fatty acid free BSA (Sigma), 500 μM carnitine (Sigma). Cells were pulsed for 4 hours with 0.5 μCi [9,10-3H(N)]-palmitic acid (Perkinelmer) and the medium was collected to analyze the released 3H2O, formed during cellular oxidation of [3H] palmitate. Briefly, medium was precipitated by 10% trichloroacetic acid (Sigma) and then supernatant was neutralized with 6N NaOH (Sigma) and loaded into ion exchange columns packed with DOWEX 1X2-400 resin (Sigma). The radioactive product was eluted with water and quantitated by liquid scintillation counting.
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