Glycosphingolipid extraction was performed with modifications from previous reports.63 (link) To each prepared membrane fraction of untreated and ManNAz-treated Caco-2 and PNT2 cells, a 500 μL solution of water : methanol : chloroform (3 : 8 : 4) was added. Samples were vortexed and centrifuged at 21 000 × g for 1 min. The supernatant was collected into a separate vial. The extraction solution was added to the remaining precipitate and the procedure repeated. Supernatants from the first and second extractions were combined. To facilitate phase separation, 100 μL of 1 M KCl was added to the solution. The upper layer was collected and dried in vacuo. GSLs were then purified by solid-phase extraction using C8 cartridges (Supelco, PA) and concentrated in vacuo prior to mass spectrometric analysis.
C8 cartridges
Manufactured by Merck Group
Sourced in Germany
The C8 cartridges are a type of lab equipment used for various analytical and purification processes. They are composed of a solid phase material with a C8 (octyl) functional group that can be used to separate, extract, or purify analytes from complex matrices. The core function of the C8 cartridges is to facilitate these types of laboratory procedures, but a more detailed description cannot be provided while maintaining an unbiased and factual approach.
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2 protocols using c8 cartridges
1
Glycosphingolipid Extraction and Purification
2
HPLC-MS Ganglioside Analysis
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High-performance liquid chromatography (HPLC)/MS grade methanol, acetic acid, and isopropyl alcohol were purchased from Sigma (St. Louis, MO, USA), and certified ACS grade chloroform came from Fisher (Fair Lawn, NJ, USA). Ammonium acetate was of analytical reagent grade and was acquired from Merck (Darmstadt, Germany), and C8 cartridges were obtained from Supelco (Bellefonte, PA). The N-omega-CD3-octadecanoyl GM3 (IS), and GM3 and GD3 ganglioside standards from buttermilk were purchased from Matreya (Pleasant Gap, PA, USA). The stock solutions of both standards were prepared separately in methanol to obtain a concentration of 1 mg/mL. Milli-Q grade water was used for all experiments.
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