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

Dette arbeidet ble støttet av et fellesskap av Akademie für Tiergesundheit (AFT) og et fellesskap fra PhD-programmet, &quot;Animal og Zoonotiske infeksjoner,&quot; ved University of Veterinary Medicine, Hannover, Tyskland, gitt til Ariane Neumann.

Samlingen av perifert blod i denne protokollen ble godkjent av lokale menneskelige forskningsetikk provisjon. Alle mennesker gitt sin skriftlig informert samtykke. 1. Isolering av human blodavledede Neutrophils ved densitetsgradientsentrifugering <ol><li> Isolering av humane, blodavledede nøytrofiler <ol><li> Lag ~ 20 ml blod på 20 ml av natrium-diatrizoat / dekstranoppløsning nær en flamme og uten blanding. </li><li> Sentrifuger i 30 minutter ved 470 x g uten brems. <...

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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. 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Metodene som beskrives her, kan brukes til å analysere bestemte lipider, så som kolesterol, ved HPTLC eller HPLC, og for å undersøke effektene av farmakologiske Lipidendringer på dannelsen av NET (se Neumann et al. 15). HPTLC er et forholdsvis kostnadseffektiv og enkel metode for å analysere et bredt spekter av lipider i et stort antall prøver. Denne metoden har vært brukt i mange forsknings- områder, inkludert antibiotika kvantifisering <sup class="...

<html> Lipider har vist seg å spille en viktig rolle i celle homeostase, celledød, host-patogen interaksjoner, og cytokin frigjørings en. Over tid har interesse for og kunnskap om virkningen av lipider i host-patogen interaksjoner eller betennelse økt, og flere publikasjoner bekrefter den sentrale rollen av visse lipider, spesielt steroid kolesterol, i cellulære responser. Farmakologisk behandling med statiner, som brukes som inhibitorer av kolesterolbiosyntese ved blokkering av 3-hydroksy-3-metylglutaryl-koenzym-A-reduktase (HMG-CoA-reduktase), kan fungere som anti-inflammatoriske midler ved å senke serumnivået av interleukin 6 og C-reaktivt protein 2. Kolesterol- og Glycosfingolipid anriket strukturer kan brukes av flere patogener, slik som bakterier og virus, som en inngangsport til verten 3, 4, 5,class = &quot;ekstern referanse&quot;&gt; 6. Sfingolipider (f.eks, sfingomyelin) er blitt vist å bli brukt av patogener for å fremme deres patogenitet 7. I makrofager, mykobakterier bruk kolesterolanriket domener for å komme inn i cellen; en reduksjon av kolesterol hemmer mycobakteriell opptak 8. Videre infeksjon av makrofager med Francisella tularensis, en zoonotisk middel ansvarlig for tularemia (også kjent som kanin feber) 9, førte til en infeksjon som ble opphevet når kolesterol ble uttømt fra membranene 10. På lignende måte ble det invasjon av vertsceller med Escherichia coli via fettrike konstruksjoner vist seg å være kolesterol-avhengige 4. Dessuten, Salmonella typhimurium smitteforsøk av epitelceller viste at kolesterol er avgjørende for patogen adgang inn i cellene 11. Kolesterol uttømming inhibited opptaket av Salmonella 11. Videre en fersk undersøkelse utført av Gilk et al. viste at cholesterol spiller en viktig rolle i opptaket av Coxiella burnetti 12. I tillegg Tuong et al. funnet at 25-Hydroxycholesterol spiller en avgjørende rolle i fagocytose av lipopolysakkarid (LPS) -stimulerte makrofager 13. Fagocytose ble redusert når makrofager ble farmakologisk behandlet for å utarme kolesterol 14. Således, kolesterol og andre lipider synes å spille en viktig rolle i inflammasjon og infeksjon, siden deres utarming kan redusere risikoen for invasjon fra flere patogener 10, 11, 12. Nylig var vi i stand til å vise at Lipidendringer, spesielt uttømming av kolesterol fra celle, indusere dannelsen av nøytrofil extracellular feller (garn) i humane, blodavledede nøytrofiler 15. Siden oppdagelsen av garn i 2004, har de vist seg å spille viktige roller i bakterie entrapment, og dermed i å hindre spredning av smitte 16, 17. NET består av en DNA-ryggrad forbundet med histoner, proteaser, og antimikrobielle peptider 16. Frigjøringen av garnene med nøytrofiler kan induseres av invaderende patogener 18, 19 og kjemiske substanser som forbol-myristat-acetat (PMA) eller statiner 16, 20. Men de detaljerte cellulære mekanismene, og spesielt den rollen av lipider i denne prosessen, er fortsatt ikke helt klart. Analysen av lipider kan føre til en bedre forståelse av de som er involvert i en rekke forskjellige cellulære prosesser og interaksjoner, for eksempel frigjøring av NET mekanismer. Cholesterol og sfingomyelin er viktige bestanddeler i cellemembranen og lipid mikrodomener, hvor de legger stabilitet og lette gruppering av de som er involvert i proteinhandel og signaleringshendelser 21 proteiner. For å undersøke den mekanistiske rolle av visse lipider, amfifile farmakologiske midler, så som cyklisk oligosakkarid metyl-β-cyklodekstrin (MβCD), kan brukes til å endre den lipidsammensetningen av en celle og for å redusere kolesterol in vitro 15. Her presenterer vi en metode for å bruke HPTLC for å analysere den lipidsammensetningen av nøytrofiler som respons på MβCD. HPLC ble brukt til å bekrefte nivået av kolesterol i nøytrofile befolkningen. Videre beskriver vi en fremgangsmåte for å visualisere dannelsen av nett ved immunofluorescens-mikroskopi i humane, blodavledede nøytrofiler som respons på 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

Lipid analyser utført ved høy-ytelses tynnsjiktkromatografi (HPTLC) er en forholdsvis enkel, kostnadseffektiv metode for å analysere et bredt spekter av lipider. Funksjonen av lipider (for eksempel i vert-patogen interaksjoner eller vert inngang) er blitt rapportert å spille en avgjørende rolle i cellulære prosesser. Her viser vi en fremgangsmåte for å bestemme lipid sammensetning, med en vekt på kolesterolnivået av primære blodavledede nøytrofiler, ved HPTLC i sammenligning med høy ytelse væskekromatografi (HPLC). Formålet var å undersøke rollen til lipid / kolesterol endringer i dannelsen av nøytrofile ekstracellulære feller (garn). NET frigivelse er kjent som en vertsforsvarsmekanisme for å hindre patogener i å spre seg inne i verten. Derfor, blodavledede humane neutrofiler ble behandlet med metyl-β-cyklodekstrin (MβCD) for å indusere lipid forandringer i cellene. Ved hjelp av HPTLC og HPLC, har vi vist at MβCD behandling av cellene fører til lipidforandringer assosiert med en signifikant reduksjon i kolesterol-innholdet i cellen. På samme tid, MβCD behandling av de nøytrofile celler førte til dannelse av garn, som vist ved immunofluorescens-mikroskopi. I sammendraget, her presenterer vi en detaljert metode for å studere lipid endringer i nøytrofile granulocytter og dannelsen av Nets.

Humane, blodavledede neutrofiler ble isolert ved tetthetsgradient sentrifugering (figur 2). For å undersøke virkningen av lipid forandringer ved neutrofiler ble cellene behandlet med 10 mM MβCD, som tømmes kolesterol fra cellen. Deretter ble lipidene isolert fra prøvene etter Bligh og Dyer (figur 1, venstre panel), som beskrevet av Brogden et al. 23. De fremstilte lipid prøvene ble applisert på silikagel HPTLC-plat...

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

Lipider er kjent for å spille en viktig rolle i cellulære funksjoner. Her beskriver vi en fremgangsmåte for å bestemme lipid sammensetningen av neutrofiler, med vekt på kolesterolnivået, ved å bruke både HPTLC og HPLC for å få en bedre forståelse av de underliggende mekanismene for nøytrofil ekstracellulær felle dannelse.

Metoder for å studere Lipidendringer av nøytrofile granulocytter og etterfølgende dannelse av Neutrofil Ekstracellulær feller

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

Forenklede menneske Nøytrofile Ekstracellulære Traps (NET) Isolasjon og håndtering

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

Høy gjennomstrømning Måling av ekstracellulært DNA Slipp og kvantitative NET-formasjonen i Human Neutrophils<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 Hjørnetann nøytrofile ekstracellulære felle formasjon: Dynamisk og kvantitativ analyse av fluorescens mikroskopi

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

Automatisert image-basert kvantifisering av nøytrofile ekstracellulære feller bruke 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

En høy gjennomstrømming analysen vurdere og kvantifisere nøytrofile ekstracellulære felle formasjon

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

Identifikasjon av nøytrofile ekstracellulære feller i parafininnebygd feline arteriell trombi ved hjelp av immunofluorescens mikroskopi

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

Morfologisk og kompositorisk analyse av nøytrofile ekstracellulære feller indusert av mikrobielle og kjemiske 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

Kvantifisering av myeloperoksidase-DNA og nøytrofile elastase-DNA-komplekser fra nøytrofile ekstracellulære feller ved hjelp av en modifisert 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

Immunfluorescensavbildning av nøytrofile ekstracellulære feller i vev fra mennesker og mus

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