Name: methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps
Uploaded: 2017-03-29T13:00:00
Description: Watch this Scientific Journal Video about Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps at JoVE.com

This work was supported by a fellowship of the Akademie f&#252;r Tiergesundheit (AfT) and a fellowship from the PhD program, "Animal and Zoonotic Infections," of the University of Veterinary Medicine, Hannover, Germany, provided to Ariane Neumann.

The collection of the peripheral blood in this protocol was approved by the local human research ethics commission. All human subjects provided their written informed consent.
1. Isolation of Human Blood-derived Neutrophils by Density Gradient Centrifugation
<ol>
	<li>Isolation of human blood-derived neutrophils
	<ol>
		<li>Layer ~20 mL of blood onto 20 mL of sodium diatrizoate/dextran solution near a flame and without mixing.</li>
		<li>Centrifuge for 30 min at 470 x g with...

<ol>
	<li>Riethmuller, J., Riehle, A., Grassme, H., Gulbins, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+rafts+in+host-pathogen+interactions.">Membrane rafts in host-pathogen interactions.</a> Biochim Biophys Acta. 1758 (12), 2139-2147 (2006).</li><li>Shahbazian, H., Atrian, A., Yazdanpanah, L., Lashkarara, G. R., Zafar Mohtashami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+effect+of+simvastatin+in+hemodialysis+patients.">Anti-inflammatory effect of simvastatin in hemodialysis patients.</a> Jundishapur J Nat Pharm Prod. 10, e17962 (2015).</li><li>Bavari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+raft+microdomains:+A+gateway+for+compartmentalized+trafficking+of+Ebola+and+Marburg+viruses.">Lipid raft microdomains: A gateway for compartmentalized trafficking of Ebola and Marburg viruses.</a> J Exp Med. 195, 593-602 (2002).</li><li>Zaas, D. W., Duncan, M., Rae Wright, ., J, S. N., Abraham, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+lipid+rafts+in+the+pathogenesis+of+bacterial+infections.">The role of lipid rafts in the pathogenesis of bacterial infections.</a> Biochim Biophys Acta. 1746 (3), 305-313 (2005).</li><li>Rohde, M., Muller, E., Chhatwal, G. S., Talay, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+cell+caveolae+act+as+an+entry-port+for+group+A+streptococci.">Host cell caveolae act as an entry-port for group A streptococci.</a> Cell Microbiol. 5 (5), 323-342 (2003).</li><li>Grassme, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+defense+against+Pseudomonas+aeruginosa+requires+ceramide-rich+membrane+rafts.">Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts.</a> Nat Med. 9 (3), 322-330 (2003).</li><li>Heung, L. J., Luberto, C., Del Poeta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+sphingolipids+in+microbial+pathogenesis.">Role of sphingolipids in microbial pathogenesis.</a> Infect Immun. 74 (1), 28-39 (2006).</li><li>Gatfield, J., Pieters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+cholesterol+in+entry+of+mycobacteria+into+macrophages.">Essential role for cholesterol in entry of mycobacteria into macrophages.</a> Science. 288 (5471), 1647-1650 (2000).</li><li>Rapini, R. P., Bolognia, J. L., Jorizzo, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dermatology 2-Volume Set. , (2007).</li><li>Tamilselvam, B., Daefler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Francisella+targets+cholesterol-rich+host+cell+membrane+domains+for+entry+into+macrophages.">Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages.</a> J Immunol. 180 (12), 8262-8271 (2008).</li><li>Garner, M. J., Hayward, R. D., Koronakis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Salmonella+pathogenicity+island+1+secretion+system+directs+cellular+cholesterol+redistribution+during+mammalian+cell+entry+and+intracellular+trafficking.">The Salmonella pathogenicity island 1 secretion system directs cellular cholesterol redistribution during mammalian cell entry and intracellular trafficking.</a> Cell Microbiol. 4 (3), 153-165 (2002).</li><li>Gilk, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+colonization+of+host+cells+in+the+absence+of+cholesterol.">Bacterial colonization of host cells in the absence of cholesterol.</a> PLoS Pathog. 9 (1), e1003107 (2013).</li><li>Tuong, Z. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ror&#945;1+and+cholesterol+25-hydroxylase+expression+attenuates+phagocytosis+in+male+Roralphasg/sg+mice.">Disruption of Ror&#945;1 and cholesterol 25-hydroxylase expression attenuates phagocytosis in male Roralphasg/sg mice.</a> Endocrinology. 154 (1), 140-149 (2013).</li><li>Bryan, A. M., Farnoud, A. M., Mor, V., Del Poeta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+cholesterol+depletion+and+its+effect+on+the+phagocytosis+of+Cryptococcus+neoformans.">Macrophage cholesterol depletion and its effect on the phagocytosis of Cryptococcus neoformans.</a> J Vis Exp. (94), (2014).</li><li>Neumann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+alterations+in+human+blood-derived+neutrophils+lead+to+formation+of+neutrophil+extracellular+traps.">Lipid alterations in human blood-derived neutrophils lead to formation of neutrophil extracellular traps.</a> Eur J Cell Biol. 93 (8-9), 347-354 (2014).</li><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>von K&#246;ckritz-Blickwede, M., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+turned+inside-out:+antimicrobial+defense+by+phagocyte+extracellular+traps.">Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps.</a> J Mol Med. 87 (8), 775-783 (2009).</li><li>Fuchs, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J. Cell Biol. 176 (2), 231-241 (2007).</li><li>Lauth, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M1+Protein+Allows+Group+A+Streptococcal+Survival+in+Phagocyte+Extracellular+Traps+through+Cathelicidin+Inhibition.">M1 Protein Allows Group A Streptococcal Survival in Phagocyte Extracellular Traps through Cathelicidin Inhibition.</a> J Innate Immun. 1 (3), 202-214 (2009).</li><li>Chow, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statins+Enhance+Formation+of+Phagocyte+Extracellular+Traps.">Statins Enhance Formation of Phagocyte Extracellular Traps.</a> Cell Host Microbe. 8 (5), 445-454 (2010).</li><li>Simons, K., Vaz, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems,+lipid+rafts,+and+cell+membranes.">Model systems, lipid rafts, and cell membranes.</a> Annu Rev Biophys Biomol Struct. 33, 269-295 (2004).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can J Biochem Physiol. 37, 911-917 (1959).</li><li>Brogden, G., Propsting, M., Adamek, M., Naim, H. Y., Steinhagen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+analysis+of+membrane+lipids+and+lipid+rafts+in+common+carp+(Cyprinus+carpio+L).">Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L).</a> Comp Biochem Physiol B Biochem Mol Biol. 169, 9-15 (2014).</li><li>Saldanha, T., Sawaya, A. C., Eberlin, M. N., Bragagnolo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+separation+and+determination+of+12+cholesterol+oxidation+products+in+fish:+comparative+study+of+RI,+UV,+and+APCI-MS+detectors.">HPLC separation and determination of 12 cholesterol oxidation products in fish: comparative study of RI, UV, and APCI-MS detectors.</a> J Agric Food Chem. 54 (12), 4107-4113 (2006).</li><li>Hubicka, U., Krzek, J., Woltynska, H., Stachacz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+identification+and+quantitative+determination+of+selected+aminoglycoside+antibiotics+by+thin-layer+chromatography+and+densitometry.">Simultaneous identification and quantitative determination of selected aminoglycoside antibiotics by thin-layer chromatography and densitometry.</a> J AOAC Int. 92 (4), 1068-1075 (2009).</li><li>Maalouf, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+lipid+composition+in+Fabry+disease+leads+to+an+intracellular+block+of+the+detergent-resistant+membrane-associated+dipeptidyl+peptidase+IV.">A modified lipid composition in Fabry disease leads to an intracellular block of the detergent-resistant membrane-associated dipeptidyl peptidase IV.</a> J Inherit Metab Dis. 33 (4), 445-449 (2010).</li><li>Correia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori's+cholesterol+uptake+impacts+resistance+to+docosahexaenoic+acid.">Helicobacter pylori's cholesterol uptake impacts resistance to docosahexaenoic acid.</a> Int J Med Microbiol. 304 (3-4), 314-320 (2014).</li><li>Gorudko, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-induced+activation+of+plasma+membrane+NADPH+oxidase+in+cholesterol-depleted+human+neutrophils.">Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils.</a> Arch Biochem Biophys. 516 (2), 173-181 (2011).</li><li>Masoud, R., Bizouarn, T., Hou&#233;e-Levin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol:+A+modulator+of+the+phagocyte+NADPH+oxidase+activity+-+A+cell-free+study.">Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study.</a> Redox Biol. 3, 16-24 (2014).</li><li>Knight, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidylarginine+deiminase+inhibition+is+immunomodulatory+and+vasculoprotective+in+murine+lupus.">Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus.</a> J Clin Invest. 123 (7), 2981-2993 (2013).</li><li>Reichel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harbour+seal+(Phoca+vitulina)+PMN+and+monocytes+release+extracellular+traps+to+capture+the+apicomplexan+parasite+Toxoplasma+gondii.">Harbour seal (Phoca vitulina) PMN and monocytes release extracellular traps to capture the apicomplexan parasite Toxoplasma gondii.</a> Dev Comp Immunol. 50 (2), 106-115 (2015).</li><li>Chuammitri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+heterophil+extracellular+traps+(HETs):+novel+defense+mechanism+of+chicken+heterophils.">Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils.</a> Vet Immunol Immunopathol. 129, 126-131 (2009).</li><li>Palic, D., Ostojic, J., Andreasen, C. B., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+cast+NETs:+Neutrophil+extracellular+traps+are+released+from+fish+neutrophils.">Fish cast NETs: Neutrophil extracellular traps are released from fish neutrophils.</a> Dev Comp Immunol. 31 (8), 805-816 (2007).</li><li>Ng, T. H., Chang, S. H., Wu, M. H., Wang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shrimp+hemocytes+release+extracellular+traps+that+kill+bacteria.">Shrimp hemocytes release extracellular traps that kill bacteria.</a> Dev Comp Immunol. 41 (4), 644-651 (2013).</li><li>Hawes, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+DNA:+the+tip+of+root+defenses?.">Extracellular DNA: the tip of root defenses?.</a> Plant Sci. 180 (6), 741-745 (2011).</li><li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+is+immunity+the+second+function+of+chromatin?.">Neutrophil extracellular traps: is immunity the second function of chromatin?.</a> J Cell Biol. 198 (5), 773-783 (2012).</li><li>Xu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+raft-associated+&#946;-adducin+is+required+for+PSGL-1-mediated+neutrophil+rolling+on+P-selectin.">Lipid raft-associated &#946;-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.</a> J Leukoc Biol. 97 (2), 297-306 (2015).</li><li>Ermert, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+neutrophil+extracellular+traps+in+microbial+infections.">Mouse neutrophil extracellular traps in microbial infections.</a> J Innate Immun. 1 (3), 181-193 (2009).</li><li>Metzler, K. D., Goosmann, C., Lubojemska, A., Zychlinsky, A., Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myeloperoxidase-containing+complex+regulates+neutrophil+elastase+release+and+actin+dynamics+during+NETosis.">A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.</a> Cell Rep. 8 (3), 883-896 (2014).</li><li>McGee, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+enhances+Helicobacter+pylori+resistance+to+antibiotics+and+LL-37.">Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37.</a> Antimicrob Agents Chemother. 55 (6), 2897-2904 (2011).</li></ol>

The methods described here can be used to analyze specific lipids, such as cholesterol, by HPTLC or HPLC and to investigate the effects of pharmacological lipid alterations on the formation of NETs (see Neumann et al.15).
HPTLC is a relatively cost-effective and simple method to analyze a broad range of lipids in a large number of samples. This method has been used in many research areas, including antibiotic quantification25, lipid stor...

Lipids have been shown to play important roles in cell homeostasis, cell death, host-pathogen interactions, and cytokine release1. Over time, interest for and knowledge on the impact of lipids in host-pathogen interactions or inflammation have increased, and several publications confirm the central role of certain lipids, especially the steroid cholesterol, in cellular responses. Pharmacological treatment with statins, which are used as inhibitors of cholesterol biosynthesis by blocking 3-hydroxy-3-methylglutaryl-coenzym-A-reductase (HMG-CoA-reductase), can act as anti-inflammatory agents by lowering the serum levels of interleukin 6 and C-reactive protein2. Cholesterol- and glycosphingolipid-enriched structures can be used by several pathogens, such as bacteria and viruses, as a gateway into the host3,4,5,6. Sphingolipids (e.g., sphingomyelin) have been shown to be used by pathogens to promote their pathogenicity7. In macrophages, mycobacteria use cholesterol-enriched domains for entering cells; a depletion of cholesterol inhibits mycobacterial uptake8. Furthermore, infection of macrophages with Francisella tularensis, a zoonotic agent responsible for tularemia (also known as rabbit fever)9, led to an infection that was abolished when cholesterol was depleted from the membranes10. Similarly, the invasion of host cells by Escherichia coli via lipid-rich structures was demonstrated to be cholesterol-dependent4. Moreover, Salmonella typhimurium infection experiments of epithelial cells demonstrated that cholesterol is essential for pathogen entry into the cells11. Cholesterol depletion inhibited the uptake of Salmonella11. Furthermore, a recent study by Gilk et al. demonstrated that cholesterol plays an important role in the uptake of Coxiella burnetti12. Additionally, Tuong et al. found that 25-hydroxycholesterol plays a crucial role in phagocytosis by lipopolysaccharide (LPS)-stimulated macrophages13. Phagocytosis was reduced when macrophages were pharmacologically treated to deplete cholesterol14. Thus, cholesterol and other lipids seem to play an important role in infection and inflammation, since their depletion can reduce the risk of invasion from several pathogens10,11,12.
Recently, we were able to show that lipid alterations, especially the depletion of cholesterol from the cell, induce the formation of neutrophil extracellular traps (NETs) in human blood-derived neutrophils15. Since the discovery of NETs in 2004, they have been shown to play critical roles in bacterial entrapment, and thus in hindering the spread of infection16,17. NETs consist of a DNA backbone associated with histones, proteases, and antimicrobial peptides16. The release of the NETs by neutrophils can be induced by invading pathogens18,19 and chemical substances such as phorbol-myristate-acetate (PMA) or statins16,20. However, the detailed cellular mechanisms, and especially the role of lipids in this process, are still not entirely clear. The analysis of lipids can lead to a better understanding of the mechanisms involved in a wide variety of cellular processes and interactions, such as the release of NETs. Cholesterol and sphingomyelin are vital constituents of the cell membrane and lipid microdomains, where they add stability and facilitate the clustering of the proteins involved in protein trafficking and signaling events21. To investigate the mechanistic role of certain lipids, amphiphilic pharmacological agents, such as the cyclic oligosaccharide methyl-&#946;-cyclodextrin (M&#946;CD), can be used to alter the lipid composition of a cell and to reduce cholesterol in vitro15. Here, we present a method to use HPTLC to analyze the lipid composition of neutrophils in response to M&#946;CD. HPLC was used to confirm the level of cholesterol in the neutrophil population. Furthermore, we describe a method to visualize the formation of NETs by immunofluorescence microscopy in human blood-derived neutrophils in response to M&#946;CD.

<table>
	<tbody>
		<tr>
			<td>Neutrophil isolation, NET staining and quantification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Flour 633 goat anti-rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-21070</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MPO&#945; antibody</td>
			<td>Dako</td>
			<td>A0398</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>3912-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Marienfeld-Neubauer improved counting chamber</td>
			<td>Celeromics</td>
			<td>MF-0640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope TCS SP5 AOBS with tandem scanner</td>
			<td>Leica</td>
			<td>DMI6000CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s PBS 10X</td>
			<td>Sigma-Aldrich</td>
			<td>P5493-1L</td>
			<td>Dilute 1:10 in water for 1X working solution</td>
		</tr>
		<tr>
			<td>Dy Light 488 conjugated highly cross-absorbed</td>
			<td>Thermo Fisher Scientific</td>
			<td>35503</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>2010</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA/Histone 1 antibody</td>
			<td>Millipore</td>
			<td>MAB3864</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td>1.8</td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>VWR</td>
			<td>630-1554</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl-&#946;-cyclodextrin</td>
			<td>Sigma-Aldrich</td>
			<td>C4555-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Carl Roth</td>
			<td>0335.3</td>
			<td>dissolve in water, heat up to 65 &#176;C and add 1N NaOH to clear solution</td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma-Aldrich</td>
			<td>P8139-1MG</td>
			<td>Stock 16 &#181;M, dissolved in 1X PBS</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymorphprep</td>
			<td>AXIS-SHIELD</td>
			<td>AN1114683</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P7481</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Reagent</td>
			<td>Invitrogen</td>
			<td>P7581</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>PAA</td>
			<td>E 15-848</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with CaCl and Mg</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypanblue</td>
			<td>Invitrogen</td>
			<td>15250-061</td>
			<td>0.4% solution</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Carl Roth</td>
			<td>3255.1</td>
			<td>endotoxin-free</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Lipid isolation and analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>33538</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;l syringe</td>
			<td>Hamilton</td>
			<td>701 NR 10 &#181;l</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>346136</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Carl Roth</td>
			<td>7336.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Canullla 26G</td>
			<td>Braun</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II)sulphatepentahydrate</td>
			<td>Merck</td>
			<td>1027805000</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Carl Roth</td>
			<td>7331.1</td>
			<td></td>
		</tr>
		<tr>
			<td>CP ATLAS software</td>
			<td>Lazarsoftware</td>
			<td>2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromolith HighResolution RP-18 endcapped 100-4.6 mm column</td>
			<td>Merck</td>
			<td>152022</td>
			<td></td>
		</tr>
		<tr>
			<td>High Performance Liquid Chromatograph Chromaster</td>
			<td>Hitachi</td>
			<td>HITA 892-0080-30Y</td>
			<td>Paramaters are dependent on individual HPLC machine</td>
		</tr>
		<tr>
			<td>HPLC UV Detector</td>
			<td>Hitachi</td>
			<td>5410</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Column Oven</td>
			<td>Hitachi</td>
			<td>5310</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Auto Sampler</td>
			<td>Hitachi</td>
			<td>5260</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Pump</td>
			<td>Hitachi</td>
			<td>5160</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>7342.1</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Hexane</td>
			<td>Carl Roth</td>
			<td>7339.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid</td>
			<td>Sigma-Aldrich</td>
			<td>30417</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>49,361,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potters</td>
			<td>LAT Garbsen</td>
			<td>5 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth</td>
			<td>CN30.3</td>
			<td></td>
		</tr>
		<tr>
			<td>HPTLC silica gel 60</td>
			<td>Merck</td>
			<td>105553</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacufuge plus basic device</td>
			<td>Eppendorf</td>
			<td>22820001</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar cell culture 48-well plate, flat bottom</td>
			<td>Sigma</td>
			<td>CLS3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Thermo Fisher Scientific</td>
			<td>1198882</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Carl Roth</td>
			<td>1879</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Tuberculin Syringe Only 1 ml</td>
			<td>BD Bioscience</td>
			<td>309659</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD 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.

Human blood-derived neutrophils were isolated by density gradient centrifugation (Figure 2). To investigate the effect of lipid alterations on neutrophils, the cells were treated with 10 mM M&#946;CD, which depletes cholesterol from the cell. Subsequently, the lipids were isolated from the samples by Bligh and Dyer (Figure 1, left panel), as described by Brogden et al.23. The prepared lipid samples were loaded onto silica ...

Watch this Scientific Journal Video about Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps at JoVE.com

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues (Video) | JoVE

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...