High-Performance Thin Layer Chromatography: A Technique for Preliminary Separation and Quantification of Lipids From Neutrophils

Published: April 30, 2023

Abstract

Source: Brogden, G., et al. Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps. J. Vis. Exp. (2017).

This video demonstrates a technique for the preliminary separation and quantification of lipids from human peripheral blood-derived neutrophils. This technique helps in the initial evaluation of target analytes before proceeding with HPLC.

Protocol

1. High-performance thin layer chromatography (HPTLC) to semi-quantify the amount of several lipids, including cholesterol, phospholipids, and sphingomyelin Prepare the following four running solutions and staining solutions. Solution 1: Mix ethyl acetate (26.6%), 1-propanol (26.6%), chloroform (26.6%), methanol (26.6%), and potassium chloride (9.6%). Prepare KCl by dissolving 0.25 g of KCl in 100 mL of HPLC-quality water. Solution 2: Mix n-hexane (73%), diethyl ether (23%), and citric …

Divulgaciones

The authors have nothing to disclose.

Materials

1-propanol Sigma-Aldrich 33538
10 µL syringe Hamilton 701 NR 10 µl
Diethyl ether Sigma-Aldrich 346136
Ethyl acetate Carl Roth 7336.2
Copper(II)sulphatepentahydrate Merck 1027805000
Chloroform Carl Roth 7331.1
CP ATLAS software Lazarsoftware 2
Methanol Carl Roth 7342.1
n-Hexane Carl Roth 7339.1
Phosphoric acid Sigma-Aldrich 30417
Potassium chloride Merck 49,361,000
HPTLC silica gel 60 Merck 105553

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High-Performance Thin Layer Chromatography: A Technique for Preliminary Separation and Quantification of Lipids From Neutrophils. J. Vis. Exp. (Pending Publication), e21105, doi: (2023).

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