L'autore corrispondente è stato finanziato dalla Fondazione di animale Morris (D15CA-907). Lo studio è stato sostenuto dall'Università di California, Davis, centro per la salute equina e centro per la salute animale compagno (2016-24-F). Gli autori desidera ringraziare Geena Ng per la sua assistenza con le figure e Nghi Nguyen per la sua assistenza con il video.

Tutti i metodi descritti qui sono stati approvati dal comitato di utilizzo presso la University of California, Davis e istituzionali cura degli animali (numero di protocollo: 18338).
1. prelievo sangue
<ol><li>Disegnare 10 mL di sangue da una vena cefalica o giugulare utilizzando un ago 21G dall'aspirazione di siringa.</li>
	<li>Per evitare un'eccessiva sollecitazione di taglio, rimuovere l'ago dalla siringa prima di trasferire il sangue in provette contenenti eparina sodica (75 USP). Capovolgere delicatamente le provette un paio di volte a garantire un'adeguata miscelazione di anticoagulante e sangue. Verifica l'evidenza di coaguli o aggregati di eritrociti prima di posizionare i campioni sul ghiaccio.</li>
</ol>2. isolamento del neutrofilo
<ol><li>Diluire il sangue anticoagulato con un volume uguale di tampone fosfato salino di gelida Dubecco (DPBS) (pH 7.4, senza cationi bivalenti). Trasferire 8 mL di sangue diluito in una provetta conica in polipropilene da 50 mL contenente 25 mL di 3% destrano (da Leuconostoc spp. sciolto in soluzione di cloruro di sodio 0,9%). Disporre il tubo verticalmente a temperatura ambiente per 30 min consentire l'aggregazione e la sedimentazione degli eritrociti.</li>
	<li>Con attenzione strato 5 mL di plasma leucocita-ricco (Figura 1) sulla cima di 5ml di media gradiente di densità in un tubo di turno-fondo 14 mL in polipropilene. Fare attenzione a non mescolare i 2 soluzioni11.</li>
	<li>Centrifugare le provette a 400 x g con accelerazione/decelerazione (A/D) impostato su 0 (nessun freno) per 30 min a temperatura ambiente.</li>
	<li>Utilizzando un 5ml pipetta sierologica raccogliere lo strato delle cellule del granulocyte bypassando il plasma e i livelli di sangue periferico (PBMC) di cellule mononucleate (Figura 1). Utilizzare una nuova pipetta ogni volta per minimizzare la contaminazione.</li>
	<li>Posto 4 a 6 mL di strato delle cellule del polymorphnuclear (PMN) in un tubo conico in polipropilene da 50 mL. Dal momento che una piccola quantità di aggregati di eritrociti può essere aspirata insieme al livello PMN, usare una pipetta sierologica da 1 mL per rimuovere delicatamente gli aggregati dell'eritrocito che sono depositati nella parte inferiore del tubo.</li>
	<li>Lisare gli eritrociti rimanenti con 20 mL di acqua distillata fredda. Capovolgere delicatamente la provetta più volte per 30 a 60 s per garantire adeguata lisi prima di aggiungere un volume uguale di soluzione di cloruro di sodio 1,8% ghiacciata.</li>
	<li>Centrifugare a 112 x g per 10 min a 4 ° C, A/D impostato a 0.</li>
	<li>Ripetere il passo di lisi degli eritrociti se questo pellet viene visualizzato in rosso. Scartare il surnatante con cura con una pipetta sierologica e delicatamente risospendere il pellet in 100 µ l di DPBS (destrosio di 5 mM, 0.9 mM MgCl2, 1,9 mM CaCl2 e 1% albumina di siero bovino, pH 7,3). Unire tutte le celle di sedimento e metterli su ghiaccio.</li>
	<li>Eseguire un conteggio delle cellule usando un analizzatore automatizzato delle cellule e verificare il conteggio di un emocitometro. Se necessario, concentrarsi PMNs su una lastra di vetro utilizzando una citocentrifuga (1.000 giri/min, 5 min) e determinare la percentuale dei neutrofili contando il numero di neutrofili rispetto al numero totale delle cellule.</li>
	<li>Determinare la fattibilità dei neutrofili eseguendo un test di esclusione del blu di trypan, come indicato dalla Strober et al. 12 isolamento successo dovrebbe produrre una redditività dal 95 al 100% e neutrofili dovrebbero essere superiori al 95%.</li>
	<li>Determinare la concentrazione dei neutrofili moltiplicando il conteggio totale delle cellule con la vitalità percentuale e la percentuale dei neutrofili. Isolamento di successo dovrebbe produrre una concentrazione dei neutrofili da 1,5 a 6 x 106/ml.</li>
	<li>Diluire i neutrofili a una concentrazione finale di 1 x 106/ml con DPBS contenente destrosio di 5 mM, 0.9 mM MgCl2, 1,9 mM di CaCl2e 1% di albumina di siero bovino con pH regolato a 7.3.</li>
</ol>3. fluorescente microscopia di neutrofili dal vivo
<ol><li>Posto da 200 a 400 μL di neutrofili (1 x 106/ml) in ciascun pozzetto della poli-L-lisina 12-pozzetti cultura piatto rivestito. Consentire i neutrofili ad aderire al fondo dei pozzetti incubando le cellule a 37 ° C per 30 min.</li>
	<li>Etichetta di acidi nucleici con 1 μM di uno dei due acidi nucleici coloranti, uno che le macchie in cellule con membrane intatte e uno che le macchie in cellule con membrane permeabili (Vedi Tabella materiali), per 10 min a temperatura ambiente.</li>
	<li>Attivare i neutrofili con il lipopolysaccharide di 100 μg/mL (LPS) (e. coli O55. B5), 100 nM phorbol 12-myristate 13-acetato (PMA) come controllo positivo, o un equivalente volume di DPBS come controllo negativo per 180 min a 37 ° C.</li>
	<li>Acquisire immagini utilizzando la GFP (eccitazione: 470/22 nm, emissione: 525/50 nm) e canali di Texas Red (eccitazione: 585/29 nm, emissione: 628/32 nm) su un microscopio a fluorescenza a 40 ingrandimenti presso i seguenti punti di tempo: 30, 90, 120 e 180 min.</li>
</ol>4. netto quantificazione e l'individuazione delle componenti netti mediante immunofluorescenza
<ol><li>Posizionare con attenzione il coprioggetto 18 mm diametro poli-D-lisina rivestito nei pozzetti di una piastra di coltura 12-pozzetti. Etichettare correttamente i pozzi.</li>
	<li>Seme 100 a 200 μL di isolato neutrofili (1 x 106/ml) direttamente sulle lamelle e consentire i neutrofili ad aderire al coprioggetto incubando le cellule a 37 ° C per 30 min.</li>
	<li>Attivare i neutrofili come descritto al punto 3.3. Inibiscono la formazione netta pretrattando i neutrofili con µΜ 200 Cl-amidine (15 min, 37 ° C) prima dell'attivazione come precedentemente descritto da Li et al. 13 assicurarsi che il controllo del proprio veicolo (DMSO) è incluso nell'esperimento.</li>
	<li>Rimuovere con cautela i vetrini con una pinzetta. Strato un foglio di pellicola di paraffina sopra un cavalletto di provetta creare pozzi poco profondi e inserire 2 o 3 gocce di 4% PFA in ogni ben10. Etichettare correttamente i pozzetti e appoggialo delicatamente con il coprioggetto upside-down sui pozzi di PFA-riempito. Consentire alle cellule di essere fissato a temperatura ambiente per 20 min.</li>
	<li>Lavare le cellule 3 volte in DPBS per 5 min, spostandoli da un pozzetto per il prossimo.</li>
	<li>Se immunolabelling non può essere eseguita subito dopo la fissazione e l'attivazione del neutrofilo, consentire vetrini coprioggetto essere essiccato inserendo i vetrini a faccia in su un pezzo di carta di assorbanza. Vetrini possono essere conservati per fino a 1 settimana a faccia in su in una piastra di coltura di 12-pozzetti etichettati a 4 ° C fino a ulteriori analisi. Lavare le cellule 3 volte in DPBS per 1 min, utilizzando la stessa tecnica come descritto in 4.4 prima di procedere al passaggio successivo.</li>
	<li>Per permeabilize le cellule, appoggiare delicatamente il vetrino coprioggetto capovolta su una goccia di 1% NP-40 per 1 min, utilizzando la tecnica descritta in 4.4. Lavare 3 volte in DPBS per 1 minuto su un agitatore meccanico.</li>
	<li>Assegnare ogni campione a un numero e utilizzare un indicatore di punte di diamante per etichetta di conseguenza i coprioggetti in modo casuale.</li>
	<li>Preparare ed etichetta individuale di Petri allineato con il film di paraffina e 100 a 200 μL di siero di capra del 5% sulla pellicola (tampone di arresto). Posare i coprioggetti upside-down sul tampone bloccante. Mettere su un rocker e incubare per 1h a 37 ° C.</li>
	<li>Lavare 3 volte in DPBS per 5 minuti su un agitatore meccanico.</li>
	<li>Diluire l'anticorpo primario (istone di 1: 400 coniglio policlonale anti-citrullinated H3 (citH3) nel 5% di siero di capra). Incubare le cellule in piastre di Petri con etichetta allineato con il film di paraffina. Appoggiare ogni vetrino coprioggetto upside-down sul 50 a 100 μL della soluzione di anticorpo diluito. Mettere su un rocker e incubare per 1h a 37° C.</li>
	<li>Lavare 3 volte in DPBS per 5 minuti su un agitatore meccanico.</li>
	<li>Diluire anticorpo secondario coniugato con un fluoroforo (1: 200) in siero di capra 5%. Incubare le cellule in piastre di Petri con etichetta allineato con il film di paraffina. Appoggiare ogni vetrino coprioggetto upside-down sul 50 a 100 μL della soluzione di anticorpo diluito. Posto su un rocker e Incubare 1h a 37 ° C al buio.</li>
	<li>Lavare 3 volte in DPBS per 5 minuti su un agitatore meccanico al buio.</li>
	<li>Per il rilevamento simultaneo del myeloperoxidase (MPO) seguire i seguenti passi per un protocollo modificato Doppio immunolabeling come descritto da Li et al. 13
</li>
	<li>Incubare le cellule in piastre di Petri con etichetta allineato con il film di paraffina. Appoggiare ogni vetrino coprioggetto upside-down su 100 e 200 μL di 10% coniglio siero sotto dolce dondolo (durante la notte, 4 ° C, al buio).</li>
	<li>Lavare 3 volte in DPBS per 5 minuti su un agitatore meccanico al buio.</li>
	<li>Incubare le cellule in capra non coniugata di 50 μg/mL anti-coniglio Fab frammenti per 2 h a temperatura ambiente su un rocker nel buio.</li>
	<li>Lavare 3 volte in DPBS per 5 minuti su un agitatore meccanico al buio.</li>
	<li>Diluire l'anticorpo primario (1: 200 coniglio anti-umani MPO anticorpi policlonali) in siero di capra 5%. Incubare le cellule in piastre di Petri con etichetta allineato con il film di paraffina. Appoggiare ogni vetrino coprioggetto upside-down sul 50 a 100 μL della soluzione di anticorpo diluito. Posto su un rocker e Incubare 1h a 37° C al buio.</li>
	<li>Diluire l'anticorpo secondario coniugato ad un fluoroforo in siero di capra del 5% e incubare come descritto in 4.8</li>
	<li>Lavare 3 volte in DPBS per 5 minuti su un agitatore meccanico al buio.</li>
	<li>Controlli di interferenza devono essere preparati da escluse incubazione con entrambi gli anticorpi primari nel secondo passaggio immunitario-etichettatura.</li>
	<li>Macchia di DNA con 300 nM di 4', 6-Diamidion-2-Phenylindole, dicloridrato (DAPI) per 5 min al buio a temperatura ambiente.</li>
	<li>Lavare 3 volte in DPBS per 1 minuto su un rocker nel buio.</li>
	<li>Applicare una goccia (~ 50 μl) del montante antifade su una lastra di vetro. Picchiettare delicatamente il bordo del coverslip su una carta assorbente per rimuovere il tampone in eccesso prima di montare il vetrino coprioggetto con le cellule upside-down. Il mezzo di montaggio dovrebbe formare uno strato sottile tra il vetrino coprioggetto e il vetrino. Per rimuovere le bolle d'aria, premere molto delicatamente il vetrino coprioggetti con una pinzetta.</li>
	<li>Permetta che i campioni curare una notte al buio a 4 ° C. Il mezzo di montaggio dovrebbe indurire per analisi microscopiche con lenti ad immersione. Conservare i campioni a 4 ° C al buio.</li>
</ol>5. del neutrofilo trappola extracellulare quantificazione
<ol><li>Accecare gli operatori di acquisire e analizzare le immagini del microscopio alle condizioni sperimentali.</li>
	<li>Usando un microscopio a fluorescenza a 40 ingrandimenti, prendere 10 immagini in modo casuale da varie regioni di ogni vetrino coprioggetti in un esperimento sotto i rispettivi canali. Assicurarsi che il tempo di esposizione, la luminosità e il contrasto di ogni canale sono mantenuti costante durante l'acquisizione di immagini.</li>
	<li>Analizzare le immagini utilizzando il software disponibile come ImageJ (NIH). Identificare le reti basate su co-localizzazione di cfDNA, citH3 e MPO (Figura 3A, B). Quantificare il numero delle reti e dei neutrofili in 10 campi casuali per ogni condizione sperimentale. Numeri di reti rilasciato può essere espressa come rapporto (numero di reti: numero totale dei neutrofili) per ogni condizione sperimentale.</li>
	<li>Per valutare gli effetti del trattamento sull'espressione intracellulare citH3, contare il numero dei neutrofili con citH3 intracellulare e il numero dei neutrofili senza citH3 intracellulare in 10 campi casuali a 40 ingrandimenti sotto i rispettivi canali ( Figura 3, frecce rosse). Espressione citH3 intracellulare può essere espresso come il rapporto tra numero dei neutrofili con citH3 intracellulare al numero di neutrofili senza citH3 intracellulare.</li>
</ol>

<ol>
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J., Radic, M. <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:+Double-edged+swords+of+innate+immunity.">Neutrophil extracellular traps: Double-edged swords of innate immunity.</a> The Journal of Immunology. 189 (6), 2689-2695 (2012).</li><li>Czaikoski, P. G., 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+Induce+Organ+Damage+during+Experimental+and+Clinical+Sepsis.">Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis.</a> PLoS One. 11 (2), e0148142 (2016).</li><li>Dwivedi, D. 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Presentiamo qui un protocollo per osservare i cambiamenti nella conformazione e cfDNA i nuclei dei neutrofili canini vivente utilizzando un colorante permeante cella sia un colorante impermeant cella. Il vantaggio principale di questo test è che permette per il rilevamento in tempo reale di formazione netta di microscopia ad alta risoluzione nei neutrofili dal vivo senza fissazione delle cellule, di conseguenza, fornendo uno strumento semplice e prezioso per l'osservazione in vitro formazione netta. Poiché que...

I neutrofili sono di breve durati granulociti responsabili della iniziale difesa contro agenti patogeni d'invasione. Neutrofili, reclutati nel sito di infezione, eliminano microrganismi di fagocitosi, la degranulazione e la generazione di ossigeno reattivo (ROS) specie1. In presenza di batteri o endotossine, neutrofili rilascio dei neutrofili extracellulare trappole (reti), composto di cromatina extracellulare decorato con gli istoni e proteine granulari come elastasi e mieloperossidasi (MPO)2. Anche se le reti sono indispensabili proprietà antimicrobiche, crescenti evidenze sperimentali e cliniche suggerisce che troppo zelante formazione netta durante la sepsi può condurre a più organo disfunzione e morte3,4, 5 , 6.
Poiché le reti possono svolgere un simile ruolo patofisiologico nei cani, gli interventi terapeutici che prevenire o diminuiscono formazione netta possono servire da nuove strategie terapeutiche in animali settici. Per questo motivo, c'è la necessità di una tecnica affidabile valutare e quantificare i componenti netti nei cani e NETosis. NET componenti tra cui DNA libero (cfDNA) e nucleosomi sono state valutate in precedenza in neutrofili canini e plasma dai clinici cani7,8,9. Facendo uso delle analisi di fluorescenza, Goggs e Letendre trovato che settici cani hanno livelli elevati di cfDNA rispetto a cani sani,8. Anche se queste tecniche sono altamente oggettiva e quantitativa, misura di cfDNA e nucleosomi come marcatori di NETosis è non specifica, poiché essi possono essere derivate da cellule necrotiche diverso da NETosing neutrofili. Qui descriviamo una tecnica che utilizza la microscopia di fluorescenza per esaminare i comportamenti dei neutrofili NETosing dal vivo. Dettagliamo anche un protocollo modificato utilizzando double-immunolabeling soggettivamente quantificare le reti e i loro componenti quali MPO e citH3 in neutrofili canini10.

<table>
	<tbody>
		<tr>
			<td>Dextran from Leuconostoc spp.</td>
			<td>Sigma</td>
			<td>31392</td>
			<td>Molecular weight 450,000 &#8211; 650,000</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Life Sciences</td>
			<td>17144002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>A1285801</td>
			<td>With divalent cations</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190136</td>
			<td>Without divalent cations</td>
		</tr>
		<tr>
			<td>E. coli O55:B5</td>
			<td>InvivoGen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Fluorescent Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S11368</td>
			<td>5 mM in DMSO; stains in cells with permeable membranes</td>
		</tr>
		<tr>
			<td>SYTO Green 16 Fluorescent Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S7578</td>
			<td>1 mM in DMSO; stains in cells with intact membranes</td>
		</tr>
		<tr>
			<td>Surface-Amps NP-40</td>
			<td>Pierce</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine coated coverslips</td>
			<td>neuVitro</td>
			<td>H-12-pdl</td>
			<td>12 mm diameter</td>
		</tr>
		<tr>
			<td>Anti-citrullinated histone H3 antibody</td>
			<td>Abcam</td>
			<td>Ab5103</td>
			<td></td>
		</tr>
		<tr>
			<td>Unconjugated goat anti-rabbit Fab fragments</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-007-003</td>
			<td>Specificity: IgG (H+L)</td>
		</tr>
		<tr>
			<td>Anti- Myeloperoxidase antibody</td>
			<td>Dako</td>
			<td>A0398</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-Diamidino-2-phenylin (DAPI)</td>
			<td>Life Technologies Corporation</td>
			<td>D1306</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy

In risposta ad agenti patogeni d'invasione, neutrofili rilasciare trappole extracellulari del neutrofilo (reti), che sono reti extracellulare del DNA decorato con gli istoni e proteine antimicrobiche. Eccessiva formazione netta (NETosis) e citH3 rilascio durante la sepsi è associato con più disfunzione dell'organo e la mortalità nei topi e nell'uomo ma le sue implicazioni nei cani sono sconosciuti. Qui, descriviamo una tecnica per isolare canini neutrofili dal sangue intero per l'osservazione e la quantificazione dei NETosis. Plasma leucocita-ricco, generato dalla sedimentazione del dextrano, è separato dai mezzi di comunicazione di commercialmente disponibile densità gradiente separazione e granulociti raccolti per conteggio e test di vitalità delle cellule. Per osservare in tempo reale NETosis dal vivo dei neutrofili, cellule permeante e macchie fluorescenti impermeant dell'acido nucleico cellulare vengono aggiunti ai neutrofili attivati da lipopolisaccaride (LPS) o phorbol 12-myristate 13-acetato (PMA). Cambiamenti nella morfologia nucleare e formazione netta sono osservati nel corso del tempo da microscopia di fluorescenza. In vitro NETosis è ulteriormente caratterizzata da co-colocalizzazione di DNA libero (cfDNA), myeloperoxidase (MPO) e citrullinato istone H3 (citH3) utilizzando un protocollo modificato di doppio-immunolabelling. Per quantificare oggettivamente formazione netta e citH3 espressione utilizzando la microscopia a fluorescenza, reti e cellule citH3-positive sono quantificate in un modo accecato utilizzando il software disponibile. Questa tecnica è un dosaggio specifico per valutare la capacità in vitro dei neutrofili canini di subire NETosis.

Usando questo protocollo dell'imaging di cellule vive, gli investigatori possono osservare la morfologia nucleare, l'integrità della membrana del plasma e la presenza di cfDNA vivente dei neutrofili. Un colorante nucleare impermeant cella macchie acidi nuclei rosso in cellule con membrane cellulari danneggiate. Un altro colorante cellulare-permeante, etichette sugli acidi nucleici intracellulare in cellule vive con le membrane intatte di plasma. Tutti i neutrofili intatti, indipendenteme...

Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

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

Descriviamo i metodi per isolare canini neutrofili dal sangue intero e visualizzare formazione netta nei neutrofili dal vivo utilizzando la microscopia a fluorescenza. Sono anche descritti protocolli per quantificare la formazione netta e citrullinato istone H3 (citH3) espressione usando la microscopia di immunofluorescenza.

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 ...

in-vitro-canine-neutrophil-extracellular-trap-formation-dynamic

Research

JoVE Journal

Immunology and Infection

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

8.6K Views.  University of California, Davis. Descriviamo i metodi per isolare canini neutrofili dal sangue intero e visualizzare formazione netta nei neutrofili dal vivo utilizzando la microscopia a fluorescenza. Sono anche descritti protocolli per quantificare la formazione netta e citrullinato istone H3 (citH3) espressione usando la microscopia di immunofluorescenza.

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

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

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Monitoraggio Crawling neutrofili intraluminale, migrazione e chemiotassi transendoteliale in tessuto al microscopio video intravitale

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In vitro</em&gt; Assay misurare Adesione neutrofili attivati ​​per primarie umane endoteliali microvascolari in condizioni statiche

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

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

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

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 ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Misurare phagosome pH Raziometrico microscopia a fluorescenza

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantificazione del fagosoma da microscopia di fluorescenza di cella singola

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

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. ...