The authors would like to acknowledge Dr. Paul Kubes for his guidance and mentoring that were central in the construction of this work.

NOTA: Tutti gli esperimenti sono stati condotti in conformità alle linee guida etiche istituzionali locali. Disegno 1. Sangue <ol><li> Attraverso venipucture antecubitale, raccogliere 14 provette di sangue da un volontario sano in top verde (eparina) tubi e capovolgere ogni tubo prima di stabilirsi sul ghiaccio. Prelevare il sangue entro 10 - 15 minuti di esperimento per garantire la resa ottimale dei neutrofili. </li></ol> 2. neutrofili Isolamento da sangue intero <ol><li> Lavare ciascun tubo verde superiore di sangue con 5 ml di PBS senza Ca e Mg per diluire il sangue. In ognuno dei 4 x 50 ml conici, aggiungere 15 ml di linfociti separazione media (LSM) a temperatura ambiente. Utilizzando un ago 18 G montato su una siringa da 60 ml, strato accuratamente il sangue diluito sul LSM, la creazione di un forte interfaccia LSM-sangue. NOTA: Evitare miscelazione degli strati più possibile. </li><li> Centrifugare a 800 xg per 30 min a 21 ° C senza interruzione. NOTA: Sudden pause puòcausare miscelazione dei diversi strati. </li><li> Osservare eritrociti e neutrofili sedimenti sul fondo. Aspirare e scartare i primi 2 strati (plasma e LSM) facendo attenzione ad eliminare l&#39;interfaccia tra questi strati che contiene linfociti e cellule mononucleate, lasciando solo lo strato rosso fondo. </li><li> Per ogni provetta, aggiungere 20 ml di tampone fosfato salino (PBS) e la soluzione di destrano 20 ml del 6%. Invertire delicatamente ogni provetta per mescolare con lo strato di eritrociti e neutrofili e lasciar riposare a temperatura ambiente per 30 min. NOTA: Questo permetterà globuli rossi (RBC) per sedimentare sul fondo. </li><li> Dopo 30 minuti, trasferire il ricco surnatante neutrofili in tubi nuovi e scartare il pellet RBC. Supernatanti centrifugare a 450 xg a 4 ° C per 5 min. Dopo centrifugazione, scartare il surnatante. Il pellet conterrà per lo più neutrofili e alcuni globuli rossi. </li><li> Preparare una soluzione di lisi aggiungendo 0,5 ml di lisante buffer per 4,5 ml di acqua sterile. Lyse restante RBC con tha preparato 5 ml di soluzione lisante, il trasferimento di tutti precipitato risospeso in un tubo. Lasciare lisi a RT al buio per 10 min. </li><li> Centrifugare a 450 xg a 4 ° C per 5 min. Gettare il surnatante e lavare pellet con 5 ml di PBS senza Ca e Mg e centrifugare di nuovo a 450 xg a 4 ° C per eliminare ogni residuo soluzione lisante. Il pellet ottenuto a questo punto contiene i neutrofili. Risospendere il pellet in 30 ml di RPMI fredda 3% e mettere in ghiaccio. NOTA: 3% RPMI è fatto integrando i media RPMI con 3% siero fetale bovino (FBS) </li><li> Verificare la purezza del campione con blu di metilene colorazione. &gt; 95% di celle deve essere granulociti con nuclei multi-lobare. Utilizzare Trypan blu colorazione per verificare&gt; 95% la vitalità delle cellule e contare la resa finale dei neutrofili. Diluire neutrofili in ghiaccio freddo 3% RPMI per ottenere una concentrazione finale di 5 x 10 6 / ml neutrofili. </li></ol> 3. NET Generation <ol><li> Stimolare neutrofili con 500 nM di PMA (ogni 30 ml di soluzione neutrofili) e incubare su un tessuto piatto piatto 150 x 25mm coltura da 20 mm di griglia per 4 ore a 37 ° C 5% CO 2. Ciò consentirà NETosis. </li><li> Dopo 4 ore di stimolazione, delicatamente aspirare e scartare i media, lasciando lo strato di NET e neutrofili aderito al fondo. Non interrompere questo strato. </li><li> Usando un totale di 15 ml di PBS freddo senza Ca e Mg per piatto, lavare il fondo di ogni piatto pipettando 15 ml di PBS sul fondo del piatto per sollevare tutto il materiale aderente dal fondo. </li><li> Raccogliere soluzione ottenuta dal lavaggio ogni piatto (passo 3.3) in un tubo da 15 ml e centrifugare per 10 min a 450 xg a 4 ° C. I neutrofili e tutte le cellule rimanenti sedimentare sul fondo, lasciando un ricco-NET supernatante privo di cellule. </li><li> Divide surnatante in provette da 1,5 ml di micro-centrifuga e centrifugare per 10 minuti a 18.000 xg a 4 ° C. Questo permetterà tutto il DNA di pellet. NOTA: La centrifugazione in grande tuBES è più facile e meno consumando se velocità così elevate sono raggiungibili in centrifuga regolare, altrimenti sarà necessario dividere supernatanti in tubi più piccoli per utilizzare ad alta velocità micro-centrifuga. </li><li> Eliminare il surnatante e risospendere tutti pellets ottenuti insieme in ghiacciata PBS ad una concentrazione corrispondente a 2 x 10 7 neutrofili per 100 ml di PBS. Ciò comporterà lo stock NET privo di cellule che può essere utilizzato per esperimenti successivi. </li><li> Misurare la concentrazione di DNA nel campione ottenuto mediante spettrofotometria o alternativo strumento quantificazione del DNA. Una concentrazione adeguata del campione deve essere compresa tra 140-180 ng / ml. </li></ol> 4. NET-Cancer Cell Static Adesione saggio <ol><li> Aggiungere 100 microlitri dello stock NET precedentemente ottenuto per pozzetto in una piastra di fondo ben piatta 96 e lasciare pozzetti cappotto O / N a 4 ° C al buio. </li><li> Tra 12 e 20 ore più tardi, verifica la formazione di un monostrato di cellule uniformiNET liberi al fondo dei pozzetti al microscopio (Figura 1). A questo punto, aspirare delicatamente tutto il materiale non aderente dalle fonti, facendo attenzione a non disturbare il monostrato NET in basso. </li><li> Aggiungere 100 ml di 1% albumina sierica bovina (BSA) soluzione bloccante in ogni pozzetto e lasciare per 1 ora a RT. </li><li> Staccare le cellule tumorali A549 da un pallone T-75 di usare 2 ml di 0,25% soluzione tripsina. Una volta rimosso, aggiungere 10 ml di mezzi A549 alle cellule trypsinized e centrifugare a 450 xg a 4 ° C per 5 min. Eliminare le cellule surnatante e risospendere in media per ottenere una concentrazione di 2 x10 4 cellule tumorali per 100 microlitri mezzi. NOTA: le cellule A549 sono state coltivate separatamente. Brevemente, le cellule sono state coltivate e mantenute in mezzi F12 DMEM contenente 10% FBS e 1% penicillina streptomicina e incubate a 37 ° C 5% CO 2. Una volta 70 - è stato raggiunto l&#39;80% di confluenza delle cellule, sono stati staccati con 0,25% tripsina-EDTA e risospese nello stesso supportodescritto sopra. </li><li> Cellule tumorali Stain utilizzando CFSE aggiungendo 1 ml di CFSE per ml di supporti e lasciare macchiare a RT per 10 min. Dopo la colorazione, centrifugare fino a 450 xg a 4 ° C per 5 min poi scarta cellule surnatante e risospendere in volume iniziale di media per ottenere una concentrazione di 2 x 10 4 cellule tumorali per 100 microlitri supporti. </li><li> Dopo 1 h di NET blocco, delicatamente aspirare soluzione bloccante e aggiungere 2 x10 4 cellule tumorali in media, che è equivalente a 100 microlitri, per ben oltre il monostrato NET e permettere ad aderire per 90 min a 37 ° C 5% CO 2. Aspirare delicatamente le cellule e aggiungere 100 ml di soluzione salina in ogni pozzetto per lavare tutte le cellule non-aderenti. </li><li> In alcuni pozzi, aggiungere 1000U di DNAse1 per pozzetto per 10 minuti prima del lavaggio a degradare NET. Questo servirà come controllo negativo. In altri pozzi, aggiungere 100 ml di acqua sterile per bene per 10 minuti, che servirà come il controllo del veicolo (VC). NOTA: 3 replica per condition sono di solito eseguite per aumentare le dimensioni del campione. </li><li> Aspirare ed eliminare tutta la soluzione nei pozzetti lasciando solo NET e cellule tumorali aderenti al fondo. Aggiungere 100 ml di soluzione di formaldeide al 4% per pozzetto per fissare le cellule tumorali aderenti alle reti e leggere analisi al microscopio a fluorescenza (Figura 1). Trama e analizzare i risultati (Figura 3). </li></ol>

<ol>
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G., Jenne, C. N., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+neutrophil+extracellular+traps+capture+bacteria+from+the+bloodstream+during+sepsis.">Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.</a> Cell Host Microbe. 12 (3), 324-333 (2012).</li><li>Pilsczek, F. 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=A+novel+mechanism+of+rapid+nuclear+neutrophil+extracellular+trap+formation+in+response+to+Staphylococcus+aureus.">A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.</a> J Immunol. 185 (12), 7413-7425 (2010).</li><li>Villanueva, E., 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=Netting+neutrophils+induce+endothelial+damage,+infiltrate+tissues,+and+expose+immunostimulatory+molecules+in+systemic+lupus+erythematosus.">Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.</a> J Immunol. 187 (1), 538-552 (2011).</li><li>Khandpur, R., 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=NETs+are+a+source+of+citrullinated+autoantigens+and+stimulate+inflammatory+responses+in+rheumatoid+arthritis.">NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.</a> Sci Transl Med. 5 (178), 178ra140 (2013).</li><li>Demers, 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=Cancers+predispose+neutrophils+to+release+extracellular+DNA+traps+that+contribute+to+cancer-associated+thrombosis.">Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.</a> Proc Natl Acad Sci U S A. 109 (32), 13076-13081 (2012).</li><li>Hahn, S., Giaglis, S., Hoesli, I., Hasler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+NETs+in+reproduction:+from+infertility+to+preeclampsia+and+the+possibility+of+fetal+loss.">Neutrophil NETs in reproduction: from infertility to preeclampsia and the possibility of fetal loss.</a> Front Immunol. 3, 362 (2012).</li><li>Cools-Lartigue, 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=Neutrophil+extracellular+traps+sequester+circulating+tumor+cells+and+promote+metastasis.">Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis.</a> J Clin Invest. , (2013).</li><li>Cools-Lartigue, J., Spicer, J., Najmeh, S., Ferri, L. <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+in+cancer+progression.">Neutrophil extracellular traps in cancer progression.</a> Cell Mol Life Sci. , (2014).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., 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:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> J Vis Exp. 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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=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></ol>

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Il protocollo di isolamento trappola neutrofili extracellulare dimostrato in questo video combina diverse tecniche utilizzate in letteratura per l&#39;isolamento dei neutrofili e la formazione NET. Semplifica un processo abbastanza complesso e variabile e offre un modo molto affidabile e replicabile per isolare NET privi di cellule purificate con meno passaggi rispetto ad altri protocolli. Inoltre, i reagenti immediatamente disponibili sono impiegati aggiungendo al protocolli semplicità e riducendo in modo significativ...

Neutrofili trappole extracellulari (NET) sono state recentemente scoperti elementi neutrofili-derivati ​​composti da filamenti di DNA extracellulare decorate da centinaia di proteine ​​e istoni. Essi sono secreti dai neutrofili nel contesto di infezione e infiammazione seguenti stimoli specifici e la loro iscrizione iniziale di far parte dei meccanismi del neutrofili di difesa contro le infezioni. Sono stati mostrati prima a promuovere la cattura di vari invasori microbici compresi batteri, virus e funghi 1-3. Trapping del microbo potrebbe poi portare alla sua distruzione sia direttamente da proteine ​​NET-associate 3,4 e indirettamente, attraverso il reclutamento locale di fagociti 5,6. Il ruolo dei NET tuttavia non sembra essere limitato a ospitare difesa. Più di recente, che hanno dimostrato di svolgere un ruolo importante nelle malattie autoimmuni 7,8, trombosi 9, i disturbi legati alla gravidanza 10 e anche la progressione del cancro11,12. Pertanto, sembra che NET svolgono un ruolo importante in un gran numero di diversi processi fisiologici. Tuttavia, data la natura romanzo di tale ricerca, molto resta da chiarire per quanto riguarda i meccanismi con cui NET esercitano i loro effetti. Qui, presentiamo un metodo semplificato per l&#39;isolamento dei NET in assenza di neutrofili. Nel seguente video, dimostriamo una tecnica semplificata e facile da isolare NET dal sangue umano intero. NET cell-free possono quindi essere usati in una serie di esperimenti in vitro compreso colorazione, imuunofluorescence, blotting nonché un certo numero di dosaggi come l&#39;adesione, la migrazione e la proliferazione. Esistono altri protocolli 13, 19, tuttavia sono generalmente lunghe, complesse, costose e spesso, bassa resa 13. Questo protocollo semplificato utilizza reagenti di base e diminuisce il numero di fasi necessarie per isolare neutrofili, minimizzando la lunghezza del Operazire massimizzando il rendimento. Il seguente metodo combina diverse tecniche per l&#39;isolamento dei neutrofili precedentemente descritti in letteratura per ottenere un semplice protocollo formare un campione molto pura di neutrofili vivi. Come suggerito in letteratura, sangue venoso viene raccolto in provette EDTA ed usata entro 10 min per evitare l&#39;attivazione dei neutrofili 14. Usiamo linfociti separazione media (LSM) gradiente di densità centrifugazione per isolare granulociti e globuli rossi dal sangue intero eparinizzato, un metodo adattato dalla tecnica di centrifugazione densità Ficoll inizialmente descritto da Boyum et al 15. LSM è una modifica della formulazione Boyum che sostituisce diatrizoato sodio per la metrizoate sodio ed è stato usato con successo in molti studi per isolare neutrofili 16-18. Dopo differenziale densità centrifugazione, monociti e linfociti vengono scartati; globuli rossi vengono poi sedimentateutilizzando una soluzione Dextran 6% 14 e le restanti globuli rossi vengono lisati di ottenere una popolazione di neutrofili puri, che si verifica con Trypan blu e blu di metilene colorazione. Molti agenti sono stati utilizzati per indurre NETosis sia in vitro che in vivo, compresi lipopolisaccaride (LPS), e forbolo miristato di acetato (PMA) e interleuchine (IL 8-) 3,5,6. Nella seguente protocollo, neutrofili isolati sono stimolati con PMA 500 ​​nM per 4 ore, che ha dimostrato di essere una concentrazione sufficiente per permettere la formazione NET coerente e affidabile senza promuovere apoptosi 19,20. Dopo l&#39;isolamento, si dimostra come NET privi di cellule ottenute da questo protocollo può essere utilizzato in un test di adesione. DNAse1 viene utilizzato per digerire e degradare NET come precedentemente descritto e quindi serve come controllo 2,3,11. Altre opzioni includono l&#39;uso di inibitori dell&#39;elastasi dei neutrofili (NEI) per inibire Formati NETon, che è un&#39;alternativa accettabile quando l&#39;obiettivo è quello di inibire la formazione NET anziché effettuare la degradazione 11. Sebbene NEi ha un certo numero di funzioni diverse, è stato precedentemente dimostrato che NET deposizione può essere inibita da NEi attraverso blocco decondensazione cromatina, degranulazione dei neutrofili nucleare e la morte 12

<table border="0" cellpadding="0" cellspacing="0" width="983">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:75px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:573px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">311-425-CL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lymphocyte Separation Medium (LSM)</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">305-010-CL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran hydrochloride (powder)</td>
			<td style="width:75px;">Spectrum</td>
			<td style="width:119px;">DE130</td>
			<td>6% Dextran solution is made by supplementing PBS with Ca and Mg with 6% Dextran powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI-1640 Medium with L-glutamine</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">350-000-CL</td>
			<td>3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BD Pharm Lyse Lysing buffer</td>
			<td style="width:75px;">BD Biosciences</td>
			<td style="width:119px;">555899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phorbol 12-myristate 13-acetate (PMA)</td>
			<td style="width:75px;">Sigma-Alrdrich</td>
			<td style="width:119px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse1</td>
			<td>Roche</td>
			<td style="width:119px;">11284932001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F12 DMEM</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">319-075-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CFSE</td>
			<td style="width:75px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Integrid tissue culture dish with 20mm grid (150 x 25mm)</td>
			<td style="width:75px;">Falcon</td>
			<td style="width:119px;">353025</td>
			<td></td>
		</tr>
	</tbody>
</table>

simplified human neutrophil extracellular traps (nets) isolation and handling

Neutrofili extracellulari Traps (NET) sono stati recentemente identificati come parte dell&#39;armamentario antimicrobica del neutrofili. Oltre al loro ruolo nella lotta contro le infezioni, una recente ricerca ha dimostrato che possono essere coinvolti in molti altri processi patologici, tra cui la progressione del cancro. Isolare NET purificati è un elemento cruciale per consentire lo studio di queste funzioni. In questo video, dimostriamo un metodo semplificato di cella di isolamento NET esenti da sangue umano intero utilizzando reagenti prontamente disponibili. Isolati NET possono poi essere utilizzati per immunofluorescenza, cancellando o vari test funzionali. Ciò consente una valutazione delle loro proprietà biologiche in assenza dei potenziali effetti confondenti di neutrofili stessi. Una tecnica di separazione gradiente di densità viene impiegato per isolare neutrofili dal donatore sano sangue intero. Isolati neutrofili sono poi stimolati da phorbol 12 MYRiState 13-acetato (PMA) per indurre NETosis. Neutrofili attivati ​​vengono poi scartati, e uno stock NET cell-free si ottiene. Abbiamo poi dimostra come NET isolato può essere utilizzato in un test di adesione sulle cellule tumorali del polmone A549 umani. Lo stock NET è usato per rivestire i pozzi di un 96 e piastra di coltura cellulare O / N, e dopo aver assicurato una adeguata formazione monostrato NET sul fondo dei pozzetti, CFSE etichettati cellule A549 sono aggiunti. Cellule aderenti sono quantificati tramite un microscopio a fluorescenza Nikon TE300. In alcuni pozzi, 1000U DNAse1 viene aggiunto 10 minuti prima di contare fino a degradare NET

Conseguimento di successo di un saggio di adesione statica con NET richiede adeguata rivestimento dei pozzetti con un monostrato di NET prima dell&#39;aggiunta di cellule tumorali (Figura 1). Tutte le fasi successive devono essere eseguiti con attenzione a non interrompere quel livello. Quando si forma un adeguato strato di NET, si prevede una consistente cellule tumorali adesione NET come si vede nella Figura 2A e 2C. Questo effetto è abolita con l&#39;aggiunta di DNA...

Watch this Scientific Journal Video about Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling at JoVE.com

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

Nella seguente protocollo, si descrive un modo molto semplice per isolare trappole neutrofili extracellulari (NET) da sangue umano intero utilizzando reagenti facilmente disponibili. Abbiamo poi mostrato come i NET isolati possono essere utilizzati in un&#39;adesione test in vitro con cellule tumorali.

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 ...

simplified-human-neutrophil-extracellular-traps-nets-isolation

Research

JoVE Journal

Immunology and Infection

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

29.7K Views.  McGill University.  Nella seguente protocollo, si descrive un modo molto semplice per isolare trappole neutrofili extracellulari (NET) da sangue umano intero utilizzando reagenti facilmente disponibili. Abbiamo poi mostrato come i NET isolati possono essere utilizzati in un'adesione test in vitro con cellule tumorali. 

Video: Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

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 intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Isolamento di cellule umane endoteliali della vena ombelicale e il loro uso nello studio della trasmigrazione dei neutrofili in condizioni di flusso

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Umano camera di flusso neutrofili saggio

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

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.

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.

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 ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Un semplice test di fluorescenza per la quantificazione del Canine neutrofili extracellulare Trappola di uscita

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualizzazione del macrofago trappole extracellulari mediante microscopia confocale

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Isolamento e caratterizzazione delle microparticelle neutrofilo per studi funzionali

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

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 histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

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

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence 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

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

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

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

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. ...