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>
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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. 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(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. 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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 authors have nothing to disclose.

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 ...

The overall goal of this method is to investigate changes in the lipid composition of neutrophils during the release of neutrophil extracellular traps by using thin layer chromatography and HPLC. This method can be used to gain more detailed information about the mechanisms that are involved in the formation of neutrophil extracellular traps, or shortly named, NET formation. The main advantage of this procedure is that it provides information on the role of lipids in the general function of neutrophils by utilizing an easy to follow protocol.The isolation of human blood-derived cells will be demonstrated by a former PhD student of our lab, Ariane Neumann. And she will be assisted by Friederike Reuner. The preparation of cells for lipid analysis will be demonstrated by Graham Brogden a post-doc in my laboratory and Diab Husein, a master student in the biochemistry program.To isolate human blood-derived neutrophils layer approximately 20 mL of blood onto 20 mL of sodium diatrizoate/dextran solution near a flame, without mixing. Centrifuge for 30 minutes at 470 times g without braking. It is critical not to mix the blood phase with the density grade in solution.After the centrifugation step plus mononuclear cells need to be carefully removed to avoid contamination or unspecific activation of cells. Remove the mononuclear cells and the yellowish plasma layer. Transfer the polymorphonuclear cell, or PMN phase, into a new 50 mL tube, and fill it up to 50 mL with 1X phosphate buffered saline.Centrifuge the sample for 10 minutes at 470 times g with braking. Then, remove the supernatant and re-suspend the pellet in 5 mL of sterile water for five seconds to lyse the erythrocytes. Immediately fill up the tube to 50 mL with 1X PBS and centrifuge the sample.Upon removing the supernatant, the pellet should be white. If it is still red, repeat these steps. Then re-suspend the pellet in 1000 microliters of Roswell Park Memorial Institute, or RPMI medium.Count the cell number using Trypan Blue staining and a hemocytometer under a light microscope. Prepare a cell suspension in RPMI at a concentration of two million cells per mL. Approximately 25 million neutrophils can be harvested from 20 mL of blood.To pharmacologically treat neutrophils for lipid analysis use 15 million of the prepared cells. Incubate the samples in the reaction tubes for two hours at 37 degrees Celsius and 5%carbon dioxide. Following centrifugation of the samples remove the supernatant, and re-suspend the cell pellet in 300 microliters of HBSS.After centrifuging the sample again using the same parameters, re-suspend the cell pellet in 1 mL of a 1:1 chloroform:methanol solution. Then homogenize the re-suspended pellet with a 26 gauge canula by pushing the sample in and out of a 1 mL syringe 10 times. To isolate the lipids from the human peripheral blood-derived neutrophils, take the prepared neutrophils and place them on ice.Pipette the neutrophils to a 15 mL screw-cap glass tube with a PTFE seal, and homogenize them by shaking for one minute. Then add 2 mL of methanol followed one minute later by 1 mL of chloroform. After shaking for another one minute, rotate the glass tubes at room temperature and 50 rpm for 30 minutes.Next, pellet the protein fraction by centrifuging the solution at 7 degrees celsius and 1, 952 times g for 10 minutes. Carefully decant the supernatant into a new 15 mL glass screw-cap tube, leaving the protein-containing pellet behind. Store the pellet at 20 degrees celsius for future quantification.Add 1 mL of chloroform and wait one minute. Then, add 1 mL of double distilled water and invert the glass screw-cap tube with the sample for 30 seconds. After centrifuging the sample as before, remove and discard the upper phase down to, but not including the cloudy layer.Dry the samples in a vacuum concentrator at 60 degrees celsius and store them at 20 degrees celsius until required. To begin, fill up to 5 mL of each solution in the corresponding glass chamber. Add any kind of filter paper to increase the running speed in each chamber.Pre-incubate a 20x10cm HPTLC silica gel 60 glass plate in the first running solution. Once the running solution reaches the top of the plate dry it for 10 minutes at 110 degrees celsius. Dissolve the lipid pellet, previously obtained after drying with a vacuum concentrator, in the desired volume of 1:1 chloroform:methanol solution, and incubate for 15 minutes at 37 degrees celsius to dissolve.Next use a ruler and a soft pencil to mark the loading spots for the desired number of samples plus at least one standard. Mark the running distance at approximately 4 cm for the first running solution and at approximately 6 cm for the second running solution. To load the samples, wash the 10 microliter syringe three times in 1:1 chloroform:methanol prior to loading each new sample.Load 10 microliters of each sample drop-wise trying to concentrate the sample on as small an area as possible. Place the plate vertically into the first chamber with running solution one. Ensure that the plate is parallel to the wall of the glass chamber to achieve a uniform migration speed.Take care that the plates are placed parallel to the back wall of the glass chambers and that the solvent fronts do not run too far to ensure that the lipids are efficiently separated. Once the solvent line has reached the first mark, remove the plate, dry it, and place it in the second solution. Repeat similar steps for the second and for the third running solutions.Leave the plate in the solution until the solvent front reaches the top of the plate. Then, remove the plate, and dry it at room temperature for one minute. Place the plate in the copper sulfate solution for seven seconds.After drying the plate thoroughly, bake it in an oven for seven minutes at 170 degrees celsius. Remove the plate from the oven and allow it to cool. To perform HPLC, attach the column to a guard cartridge on the instrument, and heat it to 32 degrees celsius.Use methanol as the mobile phase at a flow rate of 1 mL per minute and 65 bar. Set the UV detector to measure at 202 nanometers to quantify the amount of cholesterol in each sample. Wash the HPLC machine thoroughly prior to analyzing the samples and establish a cholesterol standard curve as described in the text protocol.Finally, re-suspend the samples in 500 microliters of 1:1 chloroform:methanol in amber colored 1.5 mL glass bottles with screw top red PTFE/white silicone lids before loading them on the HPLC instrument. Shown here is the lipid profile of neutrophils on an HPTLC plate. Lipids can be identified by comparing migration distances to the standard.Also shown is an HPLC chromatogram showing two large peaks. The first peak just before two minutes is chloroform. The second large peak at five minutes is cholesterol.Shown here are cholesterol standard curves determined by HPLC and by HPTLC. The standard curve obtained by HPLC is linear over a broader concentration range as compared to the HPTLC standard curve. After watching this video you should have a good understanding of how to isolate human blood-derived neutrophils and subsequently analyze their lipid composition.While attempting this procedure it is important to remember to wear nitrile gloves to protect your hands against hazardous chloroform:methanol exposure and also to work under a fume hood. Future experiments will provide further information about the mechanisms involved in NET formation and general neutrophil functions which are still not fully understood. Don't forget that working with primary human blood-derived cells requires ethical permission as well as appropriate safety precautions such as working in a respective bio-safety lab to avoid risk exposure to human pathogens.Finally the aim of our study is to find new therapeutic targets against an infection based on the innate immune system, and especially the lipid composition is very interesting for us since it will help us to understand why the neutrophil is acting antimicrobial against an infection.

methods-to-study-lipid-alterations-neutrophils-subsequent-formation

Research

JoVE Journal

Immunology and Infection

9.4K vues.  University of Veterinary Medicine Hannover. The overall goal of this method is to investigate changes in the lipid composition of neutrophils during the release of neutrophil extracellular traps by using thin layer chromatography and HPLC. This method can be used to gain more detailed information about the mechanisms that are involved in the formation of neutrophil extracellular traps, or shortly named, NET formation. The main advantage of this procedure is that it provides information on the role of lipids in the general function of n...