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).