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Methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps

Brogden, Graham; Neumann, Ariane LU ; Husein, Diab M.; Reuner, Friederike; Naim, Hassan Y. and Von Köckritz-Blickwede, Maren (2017) In Journal of Visualized Experiments 2017(121).
Abstract

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore,... (More)

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-β-cyclodextrin (MβCD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that MβCD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, MβCD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

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organization
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type
Contribution to journal
publication status
published
subject
keywords
Cholesterol, High Performance Liquid Chromatography (HPLC), High Performance Thin Layer Chromatography (HPTLC), Immunology, Issue 121, Methyl-β-cyclodextrin (MβCD), Neutrophil Extracellular Traps (NETs), Neutrophils
in
Journal of Visualized Experiments
volume
2017
issue
121
publisher
JoVE
external identifiers
  • scopus:85017235203
  • wos:000397848300004
ISSN
1940-087X
DOI
10.3791/54667
language
English
LU publication?
yes
id
30b17637-a96a-4a11-a7f1-83da9a427ff7
date added to LUP
2017-05-02 10:17:50
date last changed
2017-09-18 13:33:40
@article{30b17637-a96a-4a11-a7f1-83da9a427ff7,
  abstract     = {<p>Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-β-cyclodextrin (MβCD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that MβCD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, MβCD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.</p>},
  articleno    = {e54667},
  author       = {Brogden, Graham and Neumann, Ariane and Husein, Diab M. and Reuner, Friederike and Naim, Hassan Y. and Von Köckritz-Blickwede, Maren},
  issn         = {1940-087X},
  keyword      = {Cholesterol,High Performance Liquid Chromatography (HPLC),High Performance Thin Layer Chromatography (HPTLC),Immunology,Issue 121,Methyl-β-cyclodextrin (MβCD),Neutrophil Extracellular Traps (NETs),Neutrophils},
  language     = {eng},
  month        = {03},
  number       = {121},
  publisher    = {JoVE},
  series       = {Journal of Visualized Experiments},
  title        = {Methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps},
  url          = {http://dx.doi.org/10.3791/54667},
  volume       = {2017},
  year         = {2017},
}