Questo lavoro è stato supportato dall'istituti canadesi di ricerca sanitaria (148808 e 148792), SE è stata sostenuta da un Heart and Stroke Foundation, premio personale dall'ufficio provinciale di Ontario, March of Dimes, Ted Rogers Centre per ricerca di cuore e Peter Munk Centro cardiaco. XCC detiene un Fellowship di Banting CIHR. LA tiene un cuore & Stroke/Richard Patricia Studentship Award.

Dichiarazione etica: questo protocollo è stato esaminato e approvato dal comitato di cura degli animali alla rete di salute dell'Università (Toronto, Canada) ed è in conformità con il Consiglio canadese su Animal Care.
1. preparazione buffer
<ol><li>Preparare il tampone di HBB (di Hank bilanciato sale soluzione (HBSS), siero bovino 2% calore inattivato, 0,2% di sieroalbumina bovina). Aggiungere 10 mL di calore inattivato siero bovino e 1 g di albumina di siero bovino a 500 mL di HBSS. Filtro sterilizzare attraverso un filtro di 0,2 µm e conservare a 4 ° C.</li>
	<li>Preparare il tampone di FACS (tampone fosfato salino (PBS), inattivato con calore 2% di siero bovino, 1 mM acido etilendiamminotetraacetico (EDTA, pH 8.0)). Aggiungere inattivato con calore 10 mL siero bovino e 1 mL di EDTA 0,5 M a 500 mL di PBS. Filtro sterilizzare attraverso un filtro di 0,2 µm e conservare a 4 ° C. Nota: Soluzioni possono essere memorizzati a 4 ° C per 1 mese.</li>
</ol>2. etichettatura dei leucociti circolanti
<ol><li>Asetticamente preparare l'iniezione di CD45 anti-topo con 1 µ g di anticorpo diluito in soluzione salina sterile tamponata al fosfato (PBS) per un volume finale di iniezione di 200 µ l/mouse. Volume di carico in una siringa di insulina 28,5 G e tenere lontano dalla luce. Nota: Utilizzare due diversi fluorocromo-labelled anti-CD45 anticorpi per differenziare l'intravascolare (macchiato attraverso somministrazione endovenosa) dalla macchiatura intracardiaco (macchiato dopo digestione; Vedi punto 4).</li>
	<li>
Pre-riscaldare i topi (8 – 12 settimane vecchie) esponendo la coda ad una lampada di calore rosso per 5 min. Attenzione: Non surriscaldare l'animale. Tenere la lampada a una distanza di sicurezza (> 30 cm).<ol>
<li>Trattenere l'animale in posizione sternale utilizzando un dispositivo appropriato. Esporre e stabilizzare la coda con la mano non-dominante di immobilizzare la base della coda con l'indice e il dito medio e la regione metà distale della coda con il pollice e il dito anulare. Attenzione: Non applicare troppa forza sulla punta della coda come la pelle può essere tirata fuori. Inserire l'ago con lo smusso rivolto verso l'alto nella vena. Tenere l'ago parallelo alla vena in ogni momento, come è facile da forare la vena. Nota: Sangue entrino nella siringa può verificarsi ed è un segno di una bella iniezione. Piegare l'ago con un angolo di 90° può facilitare la somministrazione endovenosa. Confermare con il vostro ufficio di sicurezza come piegatura aghi possono rappresentare un pericolo.</li>
		<li>Iniettare per via endovenosa 200 µ l nella vena caudale. Il fluido deve fluire facilmente e temporaneamente deselezionare la vena. Attenzione: Non forzare liquido in se si incontra resistenza. Questo è segno di smarrimento dell'ago.</li>
		<li>Consentire l'anticorpo di circolare per 5 min prima umanamente eutanasia del mouse.</li>
	</ol>
</li>
</ol>3. cuore isolando e digestione
<ol><li>
Eutanasia topi usando CO2 o di altra procedura istituzionalmente accettato l'eutanasia.
	<ol>
<li>Per CO2 eutanasia, posizionare il mouse nella camera CO2 e impostare la velocità di riempimento al 20% del volume camera al minuto (cioè, se la camera è 10 L, riempimento a 2 L/min). Eseguire per 2 min e monitor mouse fino a quando la respirazione è cessato.</li>
		<li>Spegnere la CO2 e lasciare il mouse per un ulteriore 1-2 minuti verificare respirazione ha smesso prima di aprire la camera ed eseguire un pizzico di punta per confermare che il mouse è morto prima di procedere per elaborare il mouse.</li>
	</ol>
</li>
	<li>Spruzzare il mouse con etanolo al 70%. Sollevare la pelle all'addome e sezionare il mouse tagliando lungo l'inizio della linea ventrale mediana a livello dei fianchi fino allo sterno. Aprire l'addome e spostare il fegato per esporre il diaframma.</li>
	<li>
Tagliare il diaframma, quindi le costole su entrambi i lati della linea mediana, facendo attenzione ad evitare di perforare i polmoni e il cuore.
	<ol>
<li>Esporre il cuore da peeling schiena le costole, quindi primo taglio gli atrii di sinistro seguiti da atri destro. Attenzione: Usare cautela quando si tagliano gli atrii, garantendo che arterie o vene non sono ferite, come questo può ridurre l'efficienza del lavaggio il sangue contenuto negli organi.</li>
		<li>Irrorare il cuore con 15 – 20 mL di PBS freddo utilizzando una siringa da 60 mL con ago 21 G e PBS freddo. Delicatamente afferrare la base del cuore con la pinzetta e inserire l'ago nel ventricolo di sinistra vicino all'apice. Ripetere la procedura nel ventricolo di destra. Un'iniezione corretta si traduce in bianchi polmoni e fegato pallido. Nota: Perfusione deve essere eseguita entro 5 minuti dopo l'eutanasia per evitare la coagulazione del sangue, che può contaminare gli organi di interesse con cellule circolanti.</li>
	</ol>
</li>
	<li>
Tenere la base del cuore con il forcipe e delicatamente tirare il cuore, quindi staccare il cuore dal taglio delle arterie polmonari principali e vene, aorta e il sacco pericardico.
	<ol>
<li>Pulire il cuore tenendo delicatamente il cuore tra il dito indice e il pollice in un tovagliolo di carta pulito privo di lanugine e tirare fuori gli atrii ed i resti delle principali arterie e vene e qualsiasi altri contaminanti con il forcipe. Nota: Garantire la totalità degli atrii è rimosso e non altri tessuti, quali il polmone o vasi sanguigni, sono presenti come questi tessuti hanno loro proprie cellule immunitarie residente. Il polmone, in particolare, ha una popolazione di grandi cellule immuni che possa mascherare le popolazioni più piccole di quelle all'interno del miocardio.</li>
	</ol>
</li>
	<li>
Mettere il cuore in un piatto di plastica di 5cm con 50 µ l di PBS freddo.
	<ol>
<li>Tritare il cuore utilizzando forbici per dissezione curve fino a quando non ci sono nessun pezzi visibili e ha una consistenza liscia. Nota: Provare a mantenere il cuore in una area contenuta del piatto durante il tagliere per minimizzare la perdita di tessuto.</li>
		<li>Trasferimento thetrituratedheart tessuto usando il forcipe curvo in provetta da 2 mL microcentrifuga con di 800 µ l freddo Dulbecco per volta Eagle Medium (DMEM).</li>
	</ol>
</li>
	<li>
Aggiungere 450 U/mL ofcollagenase io, 60 U/mL di ialuronidasi tipo S e 60 U/mL di dnasi-I per ogni campione.
	<ol>
<li>Incubare i campioni a 37 ° C in un agitatore oscillante a 50 giri/min per 45 – 60 min.</li>
	</ol>
</li>
	<li>
Dopo la digestione, brevemente campioni di vortice (20 s) e collocare immediatamente sul ghiaccio per mantenere i campioni a 4 ° C.
	<ol>
<li>Utilizzo di una pipetta da 1 mL, pipettare campioni su e giù fino a quando i campioni di tessuto sono omogenei e non intasare la punta della pipetta. Nota: Definizione di un determinato numero di volte per pipettare può aumentare riproducibilità. Se i cuori non sono digeriti in modo ottimale, pezzi del miocardio possono ostruire la pipetta. Macinazione delicatamente la punta della pipetta contro il tubo del microcentrifuge aiuterà a omogeneizzare i grandi pezzi di tessuto.</li>
	</ol>
</li>
	<li>
Pre-bagnato un colino di cella µm 40 collocato su una provetta conica da 50 mL con 1 mL di HBB freddo.
	<ol>
<li>Aggiungere il campione omogeneizzato al filtro e lavare attraverso versando lentamente 12 – 14 mL di HBB freddo.</li>
		<li>Trasferire campioni filtrati in una provetta conica di etichettati 15ml e conservare a 4 ° C.</li>
	</ol>
</li>
	<li>
Centrifugare i campioni a 400 x g per 5 min a 4 ° C. Aspirare il sovranatante.
	<ol>
<li>Lisare cellule rosse del sangue aggiungendo 1 mL di tampone di lisi di ammonio-cloruro-potassio (ACK) per ogni campione. Risospendere i campioni nel vortex e incubare a temperatura ambiente per 5 min.</li>
		<li>Una volta completata la lisi, aggiungere 5 – 8 mL di freddo di Hank bilanciato sale soluzione (HBSS) per interrompere il hemolysis.</li>
	</ol>
</li>
	<li>Centrifugare i campioni a gper 400 x 5 min a 4 ° C. Aspirare il sovranatante.</li>
	<li>Aggiungere 1 mL di tampone di FACS per ogni campione e trasferirlo in una provetta da microcentrifuga etichettati 1,5 mL. Nota: Il protocollo può essere sospesa qui per fino a 2 h con campioni conservati a 4 ° C.</li>
</ol>4. flusso Cytometry macchiatura e analisi
<ol><li>
Centrifugare i campioni a 400 x g per 5 min a 4 ° C.
	<ol>
<li>Per ridurre il legame non specifico anticorpo, diluire 1: 100 anti-CD16/CD32 e monocito Blocker (01:20) (vedere Tabella materiali) contabilizzazione per 50 µ l FACS per campione. Incubare a temperatura ambiente per 15 min.</li>
		<li>Preparare la contabilità di diluizione (1: 250 per ogni anticorpo) anticorpo 50 µ l di FACS per campione. Nota: Un colorante di redditività può essere incluso nell'anticorpo cocktail al fine di escludere le cellule morte. In alternativa, l'esclusione di piccole dimensioni di analisi del flusso può essere usato (Figura 1C e D).</li>
		<li>Senza lavaggio, aggiungere 50 µ l di mix master anticorpo per ogni campione e mescolare.</li>
		<li>Incubare i campioni a 4 ° C per 30 min al buio.</li>
	</ol>
</li>
	<li>Aggiungere 600 µ l FACS per ciascun campione per lavare e centrifugare i campioni a 400 x g per 5 min a 4 ° C.</li>
	<li>Aspirare il supernatante e risospendere il pellet in 300 µ l FACS. Mantenere i campioni a 4 ° C al buio. Nota: Alcuni coloranti di vitalità, come DAPI, richiedono la macchiatura entro 10 min prima dell'esecuzione di citometria a flusso.</li>
	<li>Analizzare mediante citometria a flusso (Vedi figure 1 e 2) all'interno dei seguenti 3 h. Se un tempo più lungo è richiesto, a breve termine fissazione utilizzando un paraformaldeide bassa concentrazione (0,5 – 1% in PBS) è consigliato per preservare la macchiatura. Nota: Si consiglia di filtrare campioni attraverso un colino di cella di 40 µm prima dell'analisi di citometria a flusso avviare al fine di prevenire l'ostruzione della fluidica del citofluorimetro.</li>
</ol>

<ol>
	<li>Marboe, C. C., Fenoglio, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+and+natural+history+of+human+myocarditis.">Pathology and natural history of human myocarditis.</a> Pathology and Immunopathology Research. 7 (4), 226-239 (1988).</li><li>Epelman, 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=Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation.">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li><li>Pinto, A. 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=An+abundant+tissue+macrophage+population+in+the+adult+murine+heart+with+a+distinct+alternatively-activated+macrophage+profile.">An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.</a> PLoS One. 7 (5), 36814 (2012).</li><li>Lavine, K. 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=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li><li>Fujiu, K., Wang, J., Nagai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotective+function+of+cardiac+macrophages.">Cardioprotective function of cardiac macrophages.</a> Cardiovascular Research. 102 (2), 232-239 (2014).</li><li>Hulsmans, 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=Macrophages+Facilitate+Electrical+Conduction+in+the+Heart.">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Nahrendorf, 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=The+healing+myocardium+sequentially+mobilizes+two+monocyte+subsets+with+divergent+and+complementary+functions.">The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.</a> Journal of Experimental Medicine. 204 (12), 3037-3047 (2007).</li><li>Clemente-Casares, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD103(+)+Conventional+Dendritic+Cell+Surveillance+System+Prevents+Development+of+Overt+Heart+Failure+during+Subclinical+Viral+Myocarditis.">A CD103(+) Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.</a> Immunity. 47 (5), 974-989 (2017).</li><li>Van der Borght, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+Infarction+Primes+Autoreactive+T+Cells+through+Activation+of+Dendritic+Cells.">Myocardial Infarction Primes Autoreactive T Cells through Activation of Dendritic Cells.</a> Cell Reports. 18 (12), 3005-3017 (2017).</li><li>Galkina, 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=Preferential+migration+of+effector+CD8++T+cells+into+the+interstitium+of+the+normal+lung.">Preferential migration of effector CD8+ T cells into the interstitium of the normal lung.</a> Journal of Clinical Investigation. 115 (12), 3473-3483 (2005).</li><li>Edelson, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+CD103++dendritic+cells+form+a+unified+subset+developmentally+related+to+CD8alpha++conventional+dendritic+cells.">Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8alpha+ conventional dendritic cells.</a> Journal of Experimental Medicine. 207 (4), 823-836 (2010).</li></ol>

Gli autori non hanno nulla a rivelare.

Infiammazione del miocardio, o miocardite, è una caratteristica delle malattie cardiovascolari più. Tuttavia, il miocardio non è privo di propri componenti immuni negli Stati di non-malattia. Durante stato stazionario, molte cellule immunitarie risiedono nel miocardio e svolgono un ruolo essenziale di manutenzione e protezione. La caratterizzazione di queste diverse popolazioni di cellule non sarebbe stato possibile senza i metodi come quello presentato in questo protocollo.
Una combinazion...

Diverse forme di stress del miocardio o danno, inclusi ischemico (ischemia riperfusione o infarto del miocardio) e non-ischemica (ipertensione o miocardite), promuovere il reclutamento di cellule infiammatorie con riparatore e protettivo, ma anche Proprietà patogene. Già nel 1891, Romberg descritto in primo luogo la presenza di infiltrati cellulari nel miocardio dei pazienti infettati con il tifo e la scarlattina1. Tuttavia, lo studio dettagliato delle cellule immuni cardiache richiesto lo sviluppo di tecniche più avanzate di immunofenotipizzazione. Di conseguenza, solo recentemente abbiamo iniziato a capire che una popolazione eterogenea di cellule del sistema immunitario con ruoli di manutenzione essenziali durante stato stazionario risiede all'interno del miocardio.
L'istologia è stato ed è tuttora, il metodo più comune per caratterizzare le cellule immuni cardiache durante l'infiammazione. Tuttavia, mentre l'istologia rappresenta un prezioso strumento diagnostico, suo utilizzo nello studio delle cellule immuni cardiache ha limitazioni importanti. Le cellule immunitarie che risiedono nel miocardio rappresentano una percentuale molto piccola delle cellule totali e sono più o meno uniformemente distribuite in uno spazio immenso proporzionalmente. Allo stesso modo, diverse forme di infiammazione cardiaca, quali miocardite virale, mostrano modelli focali di infiammazione. Questo significa che l'analisi accurata del sistema immunitario del cuore richiedono spesso più alti gradi di campionamento istologico, l'aumento del costo per ridurre i pregiudizi. Inoltre, l'istologia fornisce una quantità molto limitata di informazioni utilizzabili nell'identificazione delle cellule (ad es. numero di parametri). Con un numero sempre crescente di sottoinsiemi di cellule che richiedono l'utilizzo di più indicatori di espressione (compresi gli indicatori di superficie, fattori di trascrizione o molecole secrete), citometria a flusso si è affermata come il più potente strumento per immunophenotyping, a causa di alto-rendimento e basso costo.
L'uso di tecniche di immunophenotyping dipende strettamente lo sviluppo di protocolli di digestione efficiente che ha permesso per l'analisi di singole cellule di un gran numero di cellule. Lo sviluppo di protocolli esaustivi di estrazione delle cellule ha aperto una nuova finestra di opportunità nello studio della immunologia cardiaco. Attraverso la combinazione di digestione, citometria a flusso e trascrittomica, le principali popolazioni di macrofagi e cellule dendritiche che risiedono nel cuore sono stati caratterizzati2,3. La popolazione più abbondante di cellule immuni cardiache durante allo stato stazionario sono CD64++ MerTK macrofagi, che possono essere ulteriormente suddivisi sulla base di loro espressione di CCR2 o CD11c2. CCR2+ macrofagi hanno origine dal midollo osseo adulto ematopoiesi, considerando che la maggior parte di CCR2 macrofagi– , che possono esprimere livelli elevati o bassi di MHC-II, sono principalmente di origine prenatale. I macrofagi hanno funzioni chiave come riparazione4 e la rimozione di detriti5 durante la ferita o sostenere connettività elettrica6 in regime stazionario. Durante diverse forme di infiammazione si verificano un importante afflusso di Ly6CCiao MHC-IIlo CD64int monociti, che più successivamente differenziarsi in Ly6CCiao CCR2+ macrofagi2,7 . Una popolazione significativamente più piccola, ma importante, di cellule che risiedono nel miocardio di individui in buona salute è composto di cellule dendritiche8,9. I due sottoinsiemi principali di cardiaca convenzionale DCs (cDCs) sono stati recentemente caratterizzati: cDC1 (CD103+ DCs) e cDC2 (CD11b+ DCs). DCs svolgono un ruolo importante nella difesa contro l'infezione8 , ma può anche promuovere la auto-ferita durante l'infiammazione, in particolare durante l'infarto miocardico9.
Qui, descriviamo un metodo semplice per l'isolamento di cellule immuni cardiache vitali dal miocardio del mouse. Il metodo combina digestione enzimatica e meccanica con una filtrazione di cella-setaccio per ottenere una sospensione di singola cellula che può essere analizzata, o ulteriormente purificata, tramite flusso cytometry ordinamento o arricchimento di biglie magnetiche. Misurazioni accurate delle cellule del miocardio extravascular richiedono aspersione cardiaca al fine di rimuovere eventuali contaminanti dal flusso sanguigno del microvasculature cardiaco. Inoltre, vi presentiamo un passaggio facoltativo di etichettatura intravascolare delle cellule immuni che possono essere utilizzate per differenziare ulteriormente le cellule del miocardio da contaminanti intravascolare, basati sul Galkina et al protocollo10. Infine, presentiamo un'analisi di citometria a flusso di base per identificare le principali sottopopolazioni macrofago e cDC.

<table border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Phosphate buffered saline</td>
			<td style="width:123px;">Wisent</td>
			<td style="width:185px;">311-010-CL</td>
			<td style="width:167px;">1x PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">21Gx 1 1/2 (0.8mmx40mm) PrecisionGlide Needle</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#8217;s Balanced Salt Solution</td>
			<td>Wisent</td>
			<td>311-511-CL</td>
			<td>1x HBSS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum</td>
			<td>Sigma</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.5M Ethylenediaminetetraacetic acid</td>
			<td>BioShop</td>
			<td>EDT111</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum filter , 0.2 &#181;m Filtropur V50</td>
			<td>Sarstedt</td>
			<td>83.1823.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">28G 1/2 1 cc insulin syringe</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">60 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td>1x DMEM with 4.5 g/L glucose and L-Glutamine and Sodium Pyruvate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>from Clostridium histolyticum</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hyaluronidase type I-S</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase-I</td>
			<td>Sigma</td>
			<td>D4513</td>
			<td>from bovine pancreas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell strainer, 40 &#181;m Nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ammonium-Chloride- Potassium (ACK) lysis buffer</td>
			<td>Lonza</td>
			<td>10-546E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 700 anti-mouse/human CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC/Cy7 anti-mouse Ly-6c</td>
			<td>Biolegend</td>
			<td>128025</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC anti-mouse CD103</td>
			<td>Biolegend</td>
			<td>121414</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Brilliant Violet 605 anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE anti-mouse Ly-6G</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pacific Blue anti-mouse I-Ab</td>
			<td>Biolegend</td>
			<td>116422</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse CD64 (FcgRI)</td>
			<td>Biolegend</td>
			<td>139315</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103113</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP/Cy5.5 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>1:40 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruStain fcX (anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">True-Stain Monocyte Blocker</td>
			<td>Biolegend</td>
			<td>426101</td>
			<td>1:20 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo V10</td>
			<td>TreeStar Inc</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse: Batf3-/-</td>
			<td>The Jackson Laboratory</td>
			<td>JAX: 013755</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and identification of extravascular immune cells of the heart

Il sistema immunitario è una componente essenziale di un cuore sano. Il miocardio è sede di una popolazione ricca dei sottoinsiemi delle cellule immuni differenti con compartimentazione funzionale sia durante stato stazionario diverse forme di infiammazione. Fino a poco tempo, lo studio delle cellule immunitarie nel cuore ha richiesto l'uso di microscopia o protocolli di digestione poco sviluppati, che fornito di sensibilità sufficiente durante l'infiammazione severa, ma erano tranquillamente in grado di identificano piccoli — ma chiave — popolazioni di cellule durante lo stato stazionario. Discutiamo qui, un metodo semplice combinazione enzimatica (collagenasi, ialuronidasi e DNasi) e digestione meccanica del cuore murino preceduta da somministrazione intravascolare di anticorpi fluorescente etichettati per differenziare piccolo ma inevitabile contaminanti intravascolare delle cellule. Questo metodo genera una sospensione di cellule vitali isolate che può essere analizzato tramite flusso cytometry per fenotipizzazione di identificazione e quantificazione, o ulteriormente purificate con fluorescenza-attivato delle cellule l'ordinamento o la separazione di biglie magnetiche per studi di analisi o in vitro trascrizionali. Includiamo un esempio di una dettagliata analisi citofluorimetrica per differenziare la chiave del macrofago e popolazioni di cellule dendritiche del cuore. Per un esperimento di dimensione medio (10 cuori) il completamento della procedura richiede 2 – 3 h.

Fin qui, nessun buon metodo è stato progettato per isolare le cellule immuni da altri componenti di cellula cardiaca, come cardiomiociti. Quindi, analisi della sospensione singola cellula cardiaca tramite flusso cytometry richiedono un pre-gating con CD45 per identificare le popolazioni delle cellule immuni, seguite dalla singola cella e piccola dimensione esclusione gating (Figura 1A). In alternativa, una vitalità colorazione possono esser...

Watch this Scientific Journal Video about Isolation and Identification of Extravascular Immune Cells of the Heart at JoVE.com

Isolation and Identification of Extravascular Immune Cells of the Heart

Isolamento e identificazione di cellule immunitarie extravascolare del cuore

Questo protocollo presenta un metodo semplice ed efficace per isolare, identificare e quantificare le cellule immuni che risiedono nel miocardio dei topi durante stato stazionario o l'infiammazione. Il protocollo combina digestione enzimatica e meccanica per la generazione di una sospensione di singola cellula che può essere ulteriormente analizzata tramite flusso cytometry.

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

isolation-identification-extravascular-immune-cells

Research

JoVE Journal

Immunology and Infection

12.5K Views.  University Health Network (UHN). Questo protocollo presenta un metodo semplice ed efficace per isolare, identificare e quantificare le cellule immuni che risiedono nel miocardio dei topi durante stato stazionario o l'infiammazione. Il protocollo combina digestione enzimatica e meccanica per la generazione di una sospensione di singola cellula che può essere ulteriormente analizzata tramite flusso cytometry.

Video: Isolation and Identification of Extravascular Immune Cells of the Heart

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Misurazione dell&#39;attività Calpain nelle celle fissi e Living in citometria a flusso

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Ricerca

Immunologia e infezioni

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sette passi per Stellate Cellule

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolamento di cellule cardiache funzionali immunitario

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

An Introduction to vespe parassite di<em&gt; Drosophila</em&gt; E la risposta immunitaria antiparassitari

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imaging intravitale del Timo mouse utilizzando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziare i ruoli funzionali di espressione genica di cellule immunitarie e non immunitarie in Colite mouse da trapianto di midollo osseo

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolamento di tessuto adiposo cellule immunitarie

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolamento e identificazione di cellule immunitarie extravascolare del cuore

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Misurazione dell'attività Calpain nelle celle fissi e Living in citometria a flusso