Oasis hlb 1cc 30 mg spe columns

Acyl-CoA measurements in primary hepatocytes were performed by liquid chromatograpy-mass spectrometry/high-resolution mass spectrometry (LC-MS/HRMS) as previously described⁴³ . Briefly, primary hepatocytes were isolated and cultured as described above in 6-well plates. At harvest, culture media was completely aspirated before harvesting cells in 0.5 mL ice-cold 10% trichloroacetic acid/well of a 6-well dish using a cell lifter. Samples were then sonicated for 10 × 0.5 second pulses to completely disrupt cellular membranes, and incubated on ice to precipitate proteins. Protein was pelleted at 16,000 × RCF for 10 minutes at 4°C. Supernatant was collected and purified by solid-phase extraction using Oasis HLB 1cc (30 mg) SPE columns (Waters). Eluate was evaporated to dryness under nitrogen gas and re-suspended in 50 μL of 5% 5-sulfosalicylic acid (w/v) for injection. Samples were analyzed by an Ultimate 3000 autosampler coupled to a Thermo Q-Exactive Plus instrument in positive electrospray ionization (ESI) mode. For isotopic tracer analysis, isotopic enrichment from [U-¹³C]-fructose (Cambridge Isotope Laboratories, CLM-1553) or [U-¹³C]-acetate (Cambridge Isotope Laboratories, CLM-440–1) was calculated to compensate for the non-linearity of isotopic enrichment using the FluxFix calculator⁴⁴ .

Acyl-CoA analyses were performed by liquid chromatograpy-mass spectrometry/high-resolution mass spectrometry (LC-MS/HRMS) as previously described (75 (link)). Briefly, approximately 20–50 ×10⁶ acinar cells were cultured in suspension in low adhesion 6 mm petri dishes. At harvest, cells were placed on ice, transferred to 15 ml falcon tubes and centrifuged at 600 xg for 2 minutes at 4 °C. Medium was aspirated and the cell pellet resuspended in 1 mL 10% (w/v) trichloroacetic acid (Sigma-Aldrich, catalog #T6399) for acyl-CoA extraction. For quantification, an equal amount (100 μL) of ¹³C₃¹⁵N₁-labeled acyl-CoA internal standard (75 (link)) was added to each sample. Samples were pulse sonicated, centrifugated and the supernatant was purified by solid-phase extraction using Oasis HLB 1cc (30 mg) SPE columns (Waters). Eluate was evaporated to dryness under nitrogen gas and re-suspended in 50 μL of 5 % 5- sulfosalicylic acid (w/v) for injection. Samples were analyzed by an Ultimate 3000 autosampler coupled to a Thermo Q-Exactive Plus instrument in positive electrospray ionization (ESI) mode. For isotopic tracer analysis, isotopic enrichment from [U-¹³C]glucose or [U-¹³C]palmitate or [U-¹³C]leucine was calculated to compensate for the non-linearity of isotopic enrichment using the FluxFix calculator (76 (link)).

Samples were thawed and kept on ice throughout processing. Cell and fraction samples in 10% (w/v) trichloroacetic acid in water were sonicated for 12 × 0.5 s pulses, protein was pelleted by centrifugation at 17,000 ×g from 10 min at 4 °C. The supernatant was purified by solid-phase extraction using Oasis HLB 1cc (30 mg) SPE columns (Waters). Columns were washed with 1 mL methanol, equilibrated with 1 mL water, loaded with supernatant, desalted with 1 mL water, and eluted with 1 mL methanol containing 25 mM ammonium acetate. The purified extracts were evaporated to dryness under nitrogen then resuspended in 55 μl 5% (w/v) 5-sulfosalicylic acid in water.
Acyl-CoAs were measured by liquid chromatography-high resolution mass spectrometry. 5–10 μl of purified samples in 5% SSA were analyzed by injection of an Ultimate 3000 Quaternary UHPLC coupled to a Q Exactive Plus (Thermo Scientific) mass spectrometer in positive ESI mode using the settings described previously (Frey et al., 2016 (link)). Quantification of acyl-CoAs was via their MS2 fragments and the targeted masses used for isotopologue analysis are indicated in Table S3. Data were integrated using Tracefinder v4.1 (Thermo Scientific) software. Isotopic enrichment in tracing experiments was calculated by normalization to unlabeled control samples using the FluxFix calculator (Trefely et al., 2016 (link)).