Varian 430 gc

After 96 h of treatment, cells were trypsinized, washed twice in PBS and resuspended in fatty acid free medium. Total lipids were extracted as described by Bligh and Dyer [27 (link)] using NaCl, methanol and choloform (Sigma-Alrich) in a 1:2:2 ratio. Thin-layer chromatography was used to separate lipid classes using silica G-plates (EMD Chemical, Gibbstown, NJ, USA) Total phospholipid fatty acids were collected into test tubes and a known amount of 17:0 standard (Avanti, Alabaster, AL, USA) was added. Fatty acids were converted to fatty acid methyl esters (FAME) by incubating for 1 h at 100 °C in hexane and boron trifluoride-methanol (Sigma-Aldrich). FAME were transferred to gas chromatography (GC) vials and samples were analyzed by GC-flame ionization detection (GC-FID) using a Varian-430 GC (Varian, Lake Forest, CA, USA) equipped with a Varian FactorFour capillary column (VF-23 ms; 30 m · 0.25 mm i.d. · 0.25 lm film thickness) and a FID. Samples were injected in splitless mode as previously described [28 (link)]. Fatty acids were identified by comparison to a reference standard consisting of GLC-68 and GLC-455 supplemented with 8:0, 10:0, and 12:0 methyl esters (Nu-Chek Prep, Elysian, MN) and quantified by comparing the area of the peaks to the 17:0 peak. Results are presented as mole percentages of total fatty acids.

A volume of 25 mL of digesta from the colon was used for pH analysis using a digital pH meter (Dostmann electronic, GmbH, Wertheim, Germany) following the manufacturer’s protocol [82 ]. The ammonia concentration was measured using an ammonia selective-ion electrode (Orion 95–12, Orion Research Inc., Franklin, MA, USA) attached to a selective ion meter (710A model, Orion Research Inc., Franklin, MA, USA). The calibration was performed using dilutions at 10, 100, and 1000 mg/L of a 0.1 M standard ammonium chloride solution. In addition, an ISA solution (Ionic Strength Adjuster, Orion Research Inc., Franklin, MA, USA) of 0.5 mL was mixed into every 25 mL sample to ensure a uniform background ionic strength [83 (link)] The final results were reported as µmol/g.
Short-chain fatty acids from the piglet colonic digesta samples were analysed in aqueous extracts by gas chromatography as described by Marin et al. [80 (link)]. Briefly, the aqueous extracts were injected into a gas chromatograph (Varian, 430-GC, Varian Inc., Palo Alto, CA, USA) after centrifugation. The GC instrument is fitted with an Elite-FFAP capillary column (inside diameter 320 mm) (Perkin Elmer, Waltham, MA, USA). Volatile fatty acids obtained from CRM46975, Supelco, Bellefonte, PA, USA, were used as standards. The final results were reported as µmol/g.