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

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Methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps. / Brogden, Graham; Neumann, Ariane; Husein, Diab M.; Reuner, Friederike; Naim, Hassan Y.; Von Köckritz-Blickwede, Maren.

I: Journal of Visualized Experiments, Vol. 2017, Nr. 121, e54667, 29.03.2017.

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Brogden, Graham ; Neumann, Ariane ; Husein, Diab M. ; Reuner, Friederike ; Naim, Hassan Y. ; Von Köckritz-Blickwede, Maren. / Methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps. I: Journal of Visualized Experiments. 2017 ; Vol. 2017, Nr. 121.

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TY - JOUR

T1 - Methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps

AU - Brogden, Graham

AU - Neumann, Ariane

AU - Husein, Diab M.

AU - Reuner, Friederike

AU - Naim, Hassan Y.

AU - Von Köckritz-Blickwede, Maren

PY - 2017/3/29

Y1 - 2017/3/29

N2 - 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.

AB - 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.

KW - Cholesterol

KW - High Performance Liquid Chromatography (HPLC)

KW - High Performance Thin Layer Chromatography (HPTLC)

KW - Immunology

KW - Issue 121

KW - Methyl-β-cyclodextrin (MβCD)

KW - Neutrophil Extracellular Traps (NETs)

KW - Neutrophils

UR - http://www.scopus.com/inward/record.url?scp=85017235203&partnerID=8YFLogxK

U2 - 10.3791/54667

DO - 10.3791/54667

M3 - Article

C2 - 28447973

AN - SCOPUS:85017235203

VL - 2017

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 121

M1 - e54667

ER -