Lipids were extracted from early 3rd instar larval fat bodies as previously described (Lam et al., 2022 (link)). The lipidomic analyses were carried out on an ExionLC-AD system coupled with a Sciex QTRAP 6500 PLUS system. The separation of individual classes of polar lipids by normal phase HPLC was carried out using a TUP-HB silica column (i.d. 150x2.1 mm, 3 μm) with the following conditions: mobile phase A (chloroform:methanol:ammonium hydroxide, 89.5:10:0.5) and mobile phase B (chloroform:methanol:ammonium hydroxide:H2O, 55:39:0.5:5.5). MRM transitions were set up for quantification by referencing spiked internal standards. The mixed internal standard includes d9-PC32:0 (16:0/16:0); d7-PE33:1 (15:0/18:1); d31-PS (d31-16:0/18:1); d7-PA33:1 (15:0/18:1); d7-PG33:1 (15:0/18:1); d7-PI33:1 (15:0/18:1); d5-CL72:8 (18:2)4; d7-LPC18:1; d7-LPE18:1; C17-LPI; C17-LPA; C17-LPS; and C17-LPG (Avanti Polar Lipids). Free fatty acids were quantitated using d31-16:0 (Sigma-Aldrich).
Qtrap 6500 plus system
Manufactured by AB Sciex
Sourced in United States
The QTRAP 6500 PLUS system is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument designed for quantitative and qualitative analysis. It features a quadrupole linear ion trap mass analyzer and can perform multiple ion monitoring (MIM) and enhanced product ion (EPI) scans.
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Lab products found in correlation
6 protocols using qtrap 6500 plus system
1
Lipidomic analysis of larval fat bodies
2
Quantitative Sugar Metabolome Analysis
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Fifty milligrams of pollen grains from just dehisced anthers were collected for sugar metabolome. The lipids were removed from the samples using the improved Bligh/Dyer extraction method (two extractions) and obtained the upper aqueous phase containing sugar and small‐molecule metabolites (Song et al., 2020 (link)). After drying with a vacuum concentrator, the samples were redissolved in 50% acetonitrile solution containing a mixture of isotope‐labelled standards. Isolation was performed on a ThermoFisher DGLC3000 using a ThermoFisher Accucore 150‐Amide HILIC (100 × 2.1 mm, 2.6 μm) column, and then quantitative analysis was carried out on the Sciex QTRAP 6500 Plus system using electrospray ionization mode by referencing to spiked internal standards including d4‐citric acid, d3‐malic acid, d4‐succinic acid, d3‐pyruvate, 7‐glucose, 13C3‐fructose, 13C12‐sucrose, 13C6‐glucose‐6‐phosphate, 13C6‐fructose‐6‐phosphate, 13C6‐fructose‐1,6‐bisphoshate, 13C12‐maltose, 13C6‐UDP‐glucose (Cambridge Isotopes Laboratory) (Shi et al., 2020 (link); Song et al., 2020 (link)). Experiments were done with four biological replicates. The source data of sugar metabolome is presented in Supplemental Data S2 .
3
Lipid Extraction and Lipidomic Analysis
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Lipids were extracted from large yellow croaker liver (YL) and rainbow trout liver (OL) (60 mg) using a modified Bligh and Dyer extraction procedure (double rounds of extraction). Lipid extracts were collected into a single tube and dried in the SpeedVac under OH mode. Samples were stored at −80 °C until processed. All lipidomic analyses were performed on an Exion LC-system coupled with a QTRAP 6500 PLUS system (Sciex, Framingham, USA), and individual lipids from various classes were quantitated as described previously63 (link).
4
Targeted Lipidomic Profiling of Epididymal WAT
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Lipids in epididymal WAT were extracted following a modified Bligh and Dyer's method described as before 26 (link). Lipid profiles were measured using a high-coverage targeted lipidomic approach constructed principally on HPLCMRM, with the modification of the selection of internal standards used for quantification. The lipidomic analyses were performed using an Exion LC-system coupled with a QTRAP 6500 PLUS system (Sciex). The content of the individual lipids from various classes were quantitated relative to the respective internal standard.
5
Quantifying Gut Fatty Acids by HPLC-MS/MS
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Short chain fatty acids (SCFAs) and medium chain fatty acids (MCFAs) in colon contents were measured using HPLC-MS/MS method as previously described (Li et al., 2019 (link)). Briefly, samples were extracted with solvent mixtures containing acetonitrile and double distilled water and analyzed on a Thermo Fisher DGLC-3000 coupled to Sciex QTRAP 6500 Plus system. Octanoic acid-1-13C1 was used as an internal standard.
6
Comprehensive Lipidomics and Gene Expression
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Lipids were isolated from tissues according to the modified Bligh and Dyer's isolation approach and dried using SpeedVac under OH mode (22) . We resuspended the lipid extracts in chloroform/methanol 1:1 (v/v) spiked with indicated internal standards. We conducted all lipidomic assessments on an Exion UPLC system coupled with a QTRAP 6500 PLUS system (Sciex) as previously documented (22, 23) . All quantification experiments used internal standard calibration (24, 25) .
Quantitative Real-Time PCR RNA was isolated from kidney cortex or cultured cells using TRIzol reagent and reversed into cDNA with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following the manufacturer's instruction. Gene expression was standardized relative to 18S ribosomal RNA or b-actin. Primers are indicated in Supplementary Table 1.
Quantitative Real-Time PCR RNA was isolated from kidney cortex or cultured cells using TRIzol reagent and reversed into cDNA with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following the manufacturer's instruction. Gene expression was standardized relative to 18S ribosomal RNA or b-actin. Primers are indicated in Supplementary Table 1.
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