Quantification of 25-hydroxycholesterol

Primary microglia were prepared and treated as described above. Media were collected and frozen at −80 °C after removing floating cells. For each sample, 5 μL of methanol or 5 μL of deuterated internal standard at a concentration of 500 ng/mL were added to 50 μL of microglia growth media separately before being mixed and then hydrolyzed using 1 N KOH at 90 °C for 2 h. The samples were then liquid-liquid extracted with methyl tert-butyl ether and the organic phase evaporated to dryness under air at 50 °C. Sample residues were reconstituted in 100 μL of 80% methanol. Reconstituted samples (5 μl) were then injected onto an Eksigent microLC 200 system. The separation was effected with a Waters Acquity 1 mm × 50 mm C18 reverse-phase column at 50 μL/min over 7 min. Data were acquired by an ABSciex QTRAP 5500 mass spectrometer using the Turbo Spray source maintained at 300 °C. Spray voltage was maintained at 4000 volts, curtain gas at 40 L/min, gas 1 at 30 L/min, and gas 2 at 30 L/min. Chromatographic peak areas of transition 385.4/367.4 (CE = 25 V, DP = 60 V) were integrated and quantified using MultiQuant 3.0 software (ABSciex).