7693a automatic liquid sampler

Lipid extracts were saponified by an addition of 1 mL of 5% HCl in MeOH. Samples were vortexed for 15 seconds then placed into a sand bath heated at 80°C for 2 hours. The resulting methanolysis reaction products were extracted with hexanes (3 X 1 mL) by vortexing for 15 seconds and then centrifuged for 4 minutes at 10,000 x g. Supernatants were transferred to new tubes and gently dried under a nitrogen stream at room temperature. Samples were resuspended in 200 µL EtOAc and transferred to GC-MS vials with insert. GC-MS analysis was performed with an Agilent 5977b GC-MS HES-MSD and an Agilent 7693A automatic liquid sampler. Samples were injected (1 µL) 10:1, with an inlet temperature of 250°C. The gas chromatograph had an initial temperature of 67°C for one minute followed by a 50°C/min ramp to 197°C, a second ramp of 10°C/minute up to 325°C began immediately with a hold time of 2 minutes. A 30-meter Agilent Zorbax DB-5MS with 10 m Duraguard capillary column was employed for chromatographic separation. Helium was used as the carrier gas at a rate of 1 mL/minute. A gain of 5.0 was used for data acquisition. High Efficiency Source and quadrupole temperatures were held at 275 °C and 190 °C, respectively.

Splenic T cells from female Ldlr^−/− and T-Abc^dkoLdlr^−/− mice fed a chow diet for 28 weeks, and young (3 months) and aged (24 months) wild-type male and female mice were isolated as described above. Cholestanol-D5 was added to the T cells as internal standard and cholesterol was extracted using hexane. For T cells from Ldlr^−/− and T-Abc^dkoLdlr^−/− mice, samples were split for measurement of either total or free cholesterol content using Gas Chromatography—Mass Spectrometry (7890B GS system, 5973 MS system, and 7693 A automatic liquid sampler from Agilent; positive chemical ionization mode with 5% ammonia in methane as reaction gas). A polar DB-WAXetr (30 m × 0.25 mm × 0.25 µm) column was used. CE content was calculated by subtracting free cholesterol from total cholesterol. Cholesterol content was normalized to cellular protein content measured by the Lowry assay. For T cells of young and aged wild-type mice, total cholesterol content was assessed using the same procedure.

Bacteriological analyses were performed by Lance Energy Services, Inc. (Houston, TX). The water sample jars were decanted and pre-filtered three times with Whatmann Filter No. 40 before analysis to remove any samples containing visible particulate matter or non-dissolved matter. The filtered water was added to a glass 20 mL scintillation vial. Conductivity was measured on a calibrated pH/CON 510 Oakton analyser (WD-35610–10) using the protocol described previously^{4 (link)}. Metal content of the produced and filtered water was analysed using ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) carried out using an Optima 4300 DV spectrophotometer analyser with an AS-93 + auto sampler⁹ . Carbon analysis was analysed using a Shimadzu TOC analyser (TOC-VCSH) using an auto-sampler (ASi-V) in TC and NPOC mode. Gas chromatography/mass spectroscopy (GC/MS) analyses were performed on an Agilent Technologies 5973 network mass selective detector with a quadrupole mass spectrometer with an Agilent Technologies 5973 network GC system, using a 30-m DB-1 capillary column (0.25-mm I.D., 0.25-μm film) and helium as the carrier gas. All samples were injected using an Agilent 7693 A Automatic Liquid Sampler with split/splitless injections of 1.0 μL, injection temperature was set at 285 °C.