Varian 430 gas chromatograph

Fatty acid methyl esters (FAME) were analyzed on a Varian-430 gas chromatograph (Varian, Lake Forest, CA) equipped with an Agilent capillary column (DB-23; 30 m × 0.25 mm i.d. × 0.25 µm film thickness, Santa Clara, CA). One microliter of FAME was injected in splitless mode. The carrier gas was helium, set to a constant flow rate of 0.7 ml/min. The injector and detector ports were set at 250 °C. FAME were eluted using a temperature program set initially at 50 °C for 2 min, followed by a ramp-up at 20 °C/min to 170 °C, and a hold at 170 °C for 1 min, and an increase of 3 °C/min to 212 °C and a hold at 212 °C for 5 min. Peaks were confirmed by identifying the retention times of authentic FAME standards of known composition (Nu-Chek-Prep, Elysian, MN, USA). Fatty-acid concentrations (nmol/g of brain) were calculated by proportional comparisons of the gas chromatography peak areas with that of the heptadecanoic acid internal standard.

A known amount of heptadecanoic acid (17:0; Nu-Check Prep, Elysian, MN) was added
to the TLE. Samples were transmethylated using 14% boron trifluoride in methanol
at 100°C for 1 h. Fatty acid methyl esters (FAMEs) were extracted with
hexane and quantified using GC-FID. FAMEs were analyzed using a Varian-430 gas
chromatograph (Varian, Lake Forest, CA) equipped with a Varian FactorFour
capillary column (VF-23ms; 30 m × 0.25 mm interior diameter × 0.25
μm film thickness) and a FID. Samples were injected in splitless mode.
The injector and detector ports were set at 250°C. FAMEs were eluted
using a temperature program set initially at 50°C for 2 min, increasing
at 20°C/min, and held at 170°C for 1 min, then increased at
3°C/min and held at 212°C for 5 min to complete the run at 32 min.
The carrier gas was helium, set to a constant flow rate of 0.7 ml/min. Peaks
were identified by retention times of authentic FAME standards (Nu-Chek Prep).
The concentration of each fatty acid was calculated by comparison with the
internal standard (17:0) (29 (link)). The
concentrations were expressed on a per gram basis then multiplied by total
weight of the tissue to determine the total amount of each fatty acid in the
tissue.

Fatty acid analyses requiring lots of material, we pooled microglia from 4 brains for n = 1. Based on our experience, we sort 400,000 cells out of the brain of P21 mice, hence measurements were made on around 1.6 million microglia. It is noted in ng/sample in the legend.
Fatty acids: Extraction and quantification of total fatty acids by GC-FID: Microglia from 3 to 4 brains per sample were lysed in 100% methanol and extracted via from the Folch method (2:1:0.8 chloroform: methanol: 0.88% KCl) and analyzed by a Varian-430 gas chromatograph (Varian, Lake Forest CA).
Lipid mediators: Isolation and quantification of brain eicosanoids and docosanoids by LC-MS-MS: Pooled microglia extracted from 3 to 4 brains per sample were lysed in 15% methanol and stored at −80 °C prior to analysis. One ng of internal standard mixture was added to each sample, and lipid mediators were extracted, analyzed by LC-MS-MS and quantified as previously described^{87 (link)}.