C17 ceramide d18 1 17 0

Total sphingolipid extract was prepared following the procedures developed in previous studies [23] (link). Briefly, 100 μl serum of NSCLC patient or healty individuals was accurately weighed into a glass bottle, in which 1.5 ml of chloroform (CHCl₃) /MeOH (1:2, v/v) and internal standard C17 Ceramide (d18:1/17:0) (Cat# 860517, Avanti Polar Lipids), sphinganine-D7 (Cat# 860658, Avanti Polar Lipids) and sphingosine1-phosphate-D7 (Cat# 860641, Avanti Polar Lipids) was added. The mixture was vortex 15 min and incubated at 48 °C for 12 h. Then, 75 μl of KOH in MeOH (1M) was added and were incubated at 37 °C for 2 h to cleave potentially interfering glycerolipids. After neutralization by formic acid and centrifuged at 3800 rpm for 8 min, the supernatant was collected and dried by nitrogen blowing (Savant SPD1010 SpeedVac Concentrator) (Thermo Fisher Scientific, USA).