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

Questo lavoro è stato sostenuto da una borsa di studio della Akademie für Tiergesundheit (AFT) e una borsa di studio del corso di dottorato, &quot;Animal e zoonotiche infezioni&quot;, dell &#39;Università di Medicina Veterinaria, Hannover, Germania, a condizione di Ariane Neumann.

La raccolta di sangue periferico in questo protocollo è stato approvato dalla commissione etica della ricerca umane locale. Tutti i soggetti umani, purché il loro consenso informato scritto. 1. Isolamento dei neutrofili Sangue umano-derivate dal gradiente di densità centrifugazione <ol><li> Isolamento di neutrofili derivati dal sangue umano <ol><li> Strato ~ 20 ml di sangue su 20 mL di sodio diatrizoato / soluzione destrano vicino a una fiamma e senza mescolare. </li><l...

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	<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. 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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. 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. 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I metodi qui descritti possono essere utilizzati per analizzare specifici lipidi, come il colesterolo, per HPTLC o HPLC e per studiare gli effetti delle alterazioni lipidiche farmacologici sulla formazione di reti (vedi Neumann et al. 15). HPTLC è un metodo relativamente economico e semplice per analizzare una vasta gamma di lipidi in un gran numero di campioni. Questo metodo è stato utilizzato in molte aree di ricerca, compresa la quantificazione antibioti...

<html> I lipidi hanno dimostrato di svolgere un ruolo importante nella omeostasi cellulare, morte cellulare, interazioni ospite-patogeno, e di citochine rilascio 1. Nel corso del tempo, l&#39;interesse e la conoscenza sull&#39;impatto dei lipidi nelle interazioni ospite-patogeno o infiammazioni sono aumentati, e diverse pubblicazioni confermano il ruolo centrale di alcuni lipidi, in particolare il colesterolo di steroidi, in risposte cellulari. trattamento farmacologico con statine, che vengono utilizzati come inibitori della biosintesi del colesterolo bloccando 3-idrossi-3-metilglutaril-Coenzima-A-reduttasi (HMG-CoA reduttasi), può agire come agenti anti-infiammatori abbassando i livelli sierici di interleuchina 6 e proteina C-reattiva 2. Colesterolo e strutture glicosfingolipidi arricchiti possono essere usate da diversi agenti patogeni, come batteri e virus, come un gateway nell&#39;ospite 3, 4, 5,class = &quot;xref&quot;&gt; 6. Sfingolipidi (ad esempio, sfingomielina) hanno dimostrato di essere utilizzati da patogeni per promuovere la loro patogenicità 7. Nei macrofagi, domini per entrare nelle cellule uso micobatteri colesterolo arricchita; una deplezione di colesterolo inibisce l&#39;assorbimento micobatterica 8. Inoltre, infezione di macrofagi con Francisella tularensis, un agente zoonotico responsabile tularemia (noto anche come febbre dei conigli) 9, ha portato ad un&#39;infezione che è stata abolita quando il colesterolo è stata esaurita dalle membrane 10. Analogamente, l&#39;invasione delle cellule ospiti di Escherichia coli tramite strutture ricche di lipidi è stata dimostrata per essere il colesterolo-dipendente 4. Inoltre, Salmonella typhimurium esperimenti di infezione di cellule epiteliali hanno dimostrato che il colesterolo è essenziale per l&#39;ingresso del patogeno nelle cellule 11. Colesterolo esaurimento inhibited l&#39;assorbimento di Salmonella 11. Inoltre, un recente studio condotto da Gilk et al. ha dimostrato che il colesterolo svolge un ruolo importante nella diffusione di Coxiella burnetti 12. Inoltre, Tuong et al. rilevato che il 25-idrossicolesterolo gioca un ruolo cruciale nella fagocitosi da lipopolisaccaride (LPS) -stimulated macrofagi 13. La fagocitosi è stato ridotto quando i macrofagi sono stati trattati farmacologicamente per esaurire il colesterolo 14. Così, il colesterolo e altri lipidi sembrano giocare un ruolo importante nella infezione e infiammazione, dal momento che il loro esaurimento può ridurre il rischio di invasione da diversi agenti patogeni 10, 11, 12. Recentemente, siamo stati in grado di dimostrare che alterazioni lipidiche, in particolare l&#39;esaurimento del colesterolo dalla cella, indurre la formazione di spazio extracellulare neutrofilir trappole (NET) in derivati dal sangue umano neutrofili 15. Dalla scoperta del NET nel 2004, essi hanno dimostrato di giocare un ruolo critico nel intrappolamento batterica, e quindi nel ostacolare la diffusione di infezioni 16, 17. NET consistono di una dorsale DNA associato con istoni, proteasi, e peptidi antimicrobici 16. Il rilascio delle reti da neutrofili può essere indotta da patogeni invasori 18, 19 e sostanze chimiche come forbolo miristato-acetato (PMA) o statine 16, 20. Tuttavia, i meccanismi cellulari dettagliati e in particolare il ruolo dei lipidi in questo processo, non sono ancora del tutto chiaro. L&#39;analisi dei lipidi può portare ad una migliore comprensione dei meccanismi coinvolti in una varietà di processi e interazioni cellulari, come ad esempio il rilascio di NET. Cholesterol e sfingomielina sono costituenti fondamentali della membrana cellulare e lipidi microdomini, dove si aggiungono la stabilità e facilitare il raggruppamento delle proteine coinvolte nel traffico di proteine e gli eventi 21 di segnalazione. Per studiare il ruolo meccanicistico di alcuni lipidi, agenti farmacologici anfifilici, come il ciclico oligosaccaride metil-β-ciclodestrina (MβCD), può essere utilizzato per modificare la composizione lipidica di una cella e per ridurre il colesterolo in vitro 15. Qui vi presentiamo un metodo per utilizzare HPTLC per analizzare la composizione lipidica dei neutrofili in risposta a MβCD. HPLC è stata utilizzata per confermare il livello di colesterolo nella popolazione dei neutrofili. Inoltre, si descrive un metodo per visualizzare la formazione di NET mediante immunofluorescenza microscopia in neutrofili derivati ​​dal sangue umano in risposta a MβCD.

<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

analisi dei lipidi eseguita da cromatografia su strato sottile ad alta prestazione (HPTLC) è un metodo relativamente semplice e conveniente di analizzare una vasta gamma di lipidi. La funzione dei lipidi (ad esempio, nelle interazioni ospite-patogeno o voce host) è stato segnalato per svolgere un ruolo cruciale nei processi cellulari. Qui mostriamo un metodo per determinare la composizione dei lipidi, con una particolare attenzione per il livello di colesterolo neutrofili emoderivati ​​primari, da HPTLC rispetto a cromatografia liquida ad alta prestazione (HPLC). L&#39;obiettivo era quello di indagare il ruolo delle alterazioni dei lipidi / colesterolo nella formazione di neutrofili trappole extracellulari (NET). rilascio NET è noto come un meccanismo di difesa dell&#39;ospite per impedire la diffusione di agenti patogeni all&#39;interno dell&#39;ospite. Pertanto, neutrofili umani derivati ​​dal sangue sono stati trattati con metil-β-ciclodestrina (MβCD) per indurre alterazioni lipidiche nelle cellule. Utilizzando HPTLC e HPLC, abbiamo dimostrato che il trattamento MβCD delle cellule porta alla lipidicaalterazioni associate con una significativa riduzione del contenuto di colesterolo della cella. Allo stesso tempo, il trattamento MβCD dei neutrofili ha portato alla formazione di reti, come dimostrato da immunofluorescenza. In sintesi, qui vi presentiamo un metodo dettagliato per studiare alterazioni lipidiche neutrofili e la formazione di reti.

Neutrofili derivati dal sangue umano sono state isolate mediante centrifugazione a gradiente di densità (Figura 2). Per studiare l&#39;effetto di alterazioni lipidiche sui neutrofili, le cellule sono state trattate con 10 mM MβCD, che esaurisce colesterolo dalla cella. Successivamente, i lipidi sono stati isolati dai campioni di Bligh e Dyer (figura 1, pannello di sinistra), come descritto da Brogden et al. 23. I campio...

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

I lipidi sono noti per svolgere un ruolo importante nelle funzioni cellulari. Qui, si descrive un metodo per determinare la composizione lipidica di neutrofili, con enfasi sul livello di colesterolo, utilizzando sia HPTLC e HPLC per ottenere una migliore comprensione dei meccanismi alla base di neutrofili extracellulare formazione trappola.

Metodi per studiare lipidiche Alterazioni nei neutrofili e la formazione successiva di neutrofili extracellulare Traps

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

Neutrofili Trappole extracellulare: Come generare e poterle visualizzare

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

Ricerca

Immunologia e infezioni

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

Semplificato umani neutrofili extracellulari Traps (NET) Isolamento e Manipolazione

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

Misura High Throughput di extracellulare DNA di uscita e quantitativa NET Formazione in neutrofili umani<em&gt; In Vitro</em

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 Vitro Canini trappola extracellulari del neutrofilo formazione: Analisi dinamica e quantitativa mediante microscopia a fluorescenza

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

Quantificazione automatizzata basata su immagini di trappole extracellulari neutrophil utilizzando 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

Un'analisi di High-throughput per valutare e quantificare la trappola extracellulari del neutrofilo formazione

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

Identificazione delle trappole extracellulari neutrophil in Paraffin-Embedded Feline Arterial Thrombi utilizzando la microscopia immunofluorescenza

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

Analisi morfologica e composizionale di trappole extracellulari neutrofile indotte da stimoli microbici e chimici

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

Quantificazione dei complessi mieloperossidasi-DNA e elastasi-DNA neutrofili da trappole extracellulari di neutrofili utilizzando un ELISA a sandwich modificato

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

Imaging in immunofluorescenza di trappole extracellulari di neutrofili in tessuti umani e murini

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