Uplc qtrap 6500 plus

The collected ileum tissues were cryopreserved in liquid nitrogen, and five samples from each group were randomly selected, then 15 samples in three groups were collected under dry ice storage and sent to Changzhou Zhongke Zhidian Biotechnology Co., Ltd., China for sphingolipid group analysis. Lipids from the samples were extracted with an improved Bligh/Dyer extraction method (two extractions). The samples were reconstituted in the isotope mixture standard, and all analyses were performed in the Electrospray Ionization (ESI) mode using an Exion UPLC-QTRAP 6500 Plus (Sciex) LC/MS instrument [24 (link)]. Phenomenex Luna silica 3 μm (inner diameter 150×2.0 mm) chromatographic column was applied to separate various polar lipids under different conditions. Mass spectrometry multiple reaction monitoring (multiple IRactionmonitoring [MRM]) was established for the identification and quantitative analysis of various lipids [25 (link), 26 (link)]. The quantification of lipid substances is carried out by the added internal standard.

For each sample, approximately 10,000 day 1 adult worms were collected and washed three times with M9 in a 15-ml tube and rotated for 30 min at room temperature to get rid of gut bacteria. After gut clearance, worms were washed twice with M9 and centrifuged. Pellets were frozen in liquid nitrogen, then kept at −80 °C until processed. Lipid extraction and liquid chromatography/mass spectrometry (LC/MS) analysis were performed by LipidALL Technologies Co., Ltd. (www.lipidall.com). Lipids were extracted twice using chloroform:methanol (1:2), with an internal standard added. LC/MS analysis was carried out by UPLC-QTRAP 6500 PLUS (Sciex). Four replicates from each condition were used for analyses. Statistical analysis was performed using one-sided Tukey’s honestly significant difference test.