Questo lavoro è stato supportato da una UNCF-Merck tesi di laurea Science Research Fellowship (JLM), National Heart, Lung, and Blood Institute concede RO1-HL-62228 (JSJ) e RO1-HL-073990 (JSJ).

1. Preparazione della soluzione Balance Hanks &#39;Salt (HBSS) <ol><li> Autoclave 1 bicchiere di vetro L, 1000 mL cilindro graduato, e mescolare bar. </li><li> Ricostituire la soluzione polvere HBSS in autoclave L 1 bicchiere di vetro per il marchio di 900 ml con acqua bidistillata. </li><li> Mescolare soluzione HBSS con 3,4 g di sale Hepes Na + e regolare il pH a un valore compreso tra 7,35-7,41 utilizzando concentrato HCL o NaOH. </li><li> Una volta che il pH desiderato è stato raggiunto e rimane stabile, aggiungere 5 g di BSA per la soluzione a temperatura ambiente fino a quando la albumina è completamente sciolto. </li><li> Aggiungere 10 ml di acqua antibiotico, antimyotic, alla soluzione e bidistillata fino a quando il volume totale è di 1000 ml marchio. </li><li> Mescolare bene la soluzione. </li><li> Filtrare la soluzione HBSS e separare in tubi Falcon da 50 ml per un uso successivo. Tubi aggiuntivi possono essere congelati e utilizzati in un secondo momento. </li></ol> 2. Animal PREPARAZIOn - Inserimento del tubo tracheale <ol><li> Preparare un vassoio con strumenti e materiali necessari per l&#39;inserimento di un tubo tracheale (ad esempio pinze, forbici, tubo tracheale, 3-0 sutura nera intrecciata, ventilatore per piccoli animali, ecc.) </li><li> Una volta che il ratto è completamente anestetizzato con un cocktail di ketamina / xylazina nel rapporto di 2 a 1 xylazina ketamina, radere l&#39;addome, torace e gola del ratto. Per garantire che un profondo stato anestetico è stato raggiunto, pizzicare piedi del ratto e controllare per il ritiro pedale dell&#39;arto. Assicurarsi che il ratto è profondamente anestetizzati prima di fare tutte le incisioni. </li><li> Posizionare il topo con la testa sollevata su un tagliere che è inclinato di 30 ° di inclinazione </li><li> Eseguire un&#39;incisione cutanea a partire dalla metà dell&#39;addome al di sotto della mandibola. Sezionare via i muscoli del triangolo anteriore del collo per esporre la trachea. </li><li> Posizionare un 3-0 sutura nero intrecciato con la trachea. </li><li> Fare un&#39;incisione nella trachea tra gli anelli cartilaginei unod inserire il tubo tracheale. Legare saldamente la sutura per fissare il tubo tracheale per la trachea. </li><li> Collegare il tubo tracheale di un ventilatore piccolo animale. </li></ol> 3. Preparazione dello strumento per l&#39;isolamento delle cellule immunitarie <ol><li> Preparare un vassoio con gli strumenti per la procedura di isolamento delle cellule immunitarie (ad esempio pinze, forbici, HBSS, tagliato morbido teflon 24 gauge Baxter intravascolare sopra l&#39;ago rapido cath) </li><li> Preparare due siringhe 10cc, riempire una siringa con HBSS per introdurre il buffer nel sacco pericardico e prenotare l&#39;altro per l&#39;aspirazione delle celle contenenti HBSS. 3.2.1. Ridurre la punta in Teflon del calibro 24 Baxter intravascolare nel corso del rapido-cath ago (1,6 cm) da 1 / 2 a 3 / 4 della sua lunghezza originale per la procedura di isolamento. </li><li> Mettere tutte le siringhe, tamponi, e un vuoto da 15 ml tubo Falcon (per tenere il liquido estratto, una volta procedura è terminata) su ghiaccio. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Cella di isolamento immunitario <ol><li> Fai una incisione sulla linea mediana della parete addominale (lungo la linea alba) fino a raggiungere il processo xifoideo. </li><li> Partendo un po &#39;lateralmente al processo xifoideo, fare incisioni bilaterali attraverso la gabbia toracica continua con un angolo verso l&#39;ascella. Fare attenzione a non forare i polmoni, il cuore o pericardio. </li><li> Una volta che le incisioni su entrambi i lati dello sterno sono completi, sezionare via la membrana dal costole inferiori, il taglio laterale a mediale. Nota: Siate sempre consapevoli di dove il pericardio si collega al diaframma e lo sterno per evitare danni. Utilizzare il legamento falciforme come punto di riferimento per notare dove il pericardio è attaccato sul lato opposto della membrana. Inoltre, se il pericardio è tagliato, la capacità di isolare le cellule si perde. </li><li> Sollevare delicatamente un lato della gabbia toracica per esporre la cavità toracica e il cuore (ancora incapsulato dal pericardio).</li><li> Prendere la siringa riempita HBSS e trovare un punto nel sacco pericardico circa a metà strada tra lo sterno e il cuore (preferibilmente più rostrale rispetto caudale) e inserire la punta del Teflon accorciato rapido cath attraverso entrambi gli strati del pericardio. </li><li> Una volta che il catetere è a posto, procedere con delicatezza riempire lo spazio pericardico con 2 o 3 ml di tampone HBBS. Saprete immediatamente se il catetere è posizionato correttamente quando si nota lo spazio pericardico espansione. Se nessun riempimento è testimoniata, prelevare e riposizionare il rapido-cath. </li><li> Quando il sacco pericardico è pieno, prendere l&#39;altra siringa da 10 cc e posizionare la punta indietro nel pericardio e iniziare aspirare il tampone. Il foro di entrata lo stesso può essere usato o un altro possono essere creati. Assicurarsi che la punta non arrivare troppo a ridosso delle mura del sacco pericardico, fornendo aspirazione, soprattutto sul lato inferiore del cuore. Ciò potrebbe portare a pungere il pericardio e la perdita di cellule contenenti cellules. </li><li> Mentre aspirare il tampone, la punta del catetere può essere spostato all&#39;interno del sacco pericardico al fine di raccogliere tampone quanto più possibile. </li></ol> Nota: se nel farlo leggermente strappare il pericardio e fare il buco nella parte superiore del cuore più grande del foro originale, non fatevi prendere dal panico. Finché il pericardio circonda ancora abbastanza del cuore è ancora possibile eseguire un&#39;altra lavata sopra il cuore anche se con una minore quantità di tampone. Per la raccolta di una quantità ottimale di cellule, 3-4 lavaggi sono necessari ulteriori. <ol start="9"><li> Posizionare il tampone estratto nei tubi Falcon che sono stati immessi sul ghiaccio. </li><li> Rimuovere cuore, polmoni, e / o qualsiasi altro tessuto, se necessario, per ulteriori studi. Dopo la raccolta delle cellule immunitarie e di altri tessuti, il topo deve essere rapidamente e umanamente eutanasia. </li><li> Centrifugare i campioni raccolti cellule immunitarie per 10 min a 200 gr a 4 ° C. Tenere sempre i tubi in ghiaccioquando non in uso. </li><li> Rimuovere il surnatante e ricostituire il pellet in 1 ml di tampone Hyclone o HBSS. </li></ol> Nota: è importante contare il numero di cellule immunitarie ottenute da ogni cuore dopo la procedura di isolamento delle cellule immunitarie. 5. Rappresentante dei risultati: Un esempio di isolato mastociti cardiaci ottenuti con questa tecnica e colorati con blu di toluidina e safranina O / alcian blu sono presentati nelle figure 2A e 2B, rispettivamente. Come si può vedere in figura 3, L&#39;albero cardiaco, hanno mantenuto la loro capacità di rilascio di istamina dopo il trattamento con attivatori noto come 48/80 composto e sostanza P, come descritto nella Morgan et al. 9 appezzamenti Rappresentante dispersione di citometria a flusso delle cellule CD4 + e cellule CD8 + sono presentati nelle figure 4 e 5, rispettivamente. I linfociti T sono ulteriormente identificati e fenotipizzati come una popolazione di cellule da questa tecnica. Figura 1. Diagramma di flusso della procedura di isolamento delle cellule cardiache immunitario. <img alt="Figura 2" src="/files/ftp_upload/3020/3020fig2.jpg" /> . Immagini rappresentative Figura 2 istologica dei mastociti cardiaci sono macchiati come segue: (A) con blu di toluidina e (B) sono safranina O / Alcian blu. <img alt="Figura 3" src="/files/ftp_upload/3020/3020fig3.jpg" /> Figura 3. Studio funzionale dei mastociti cardiaci isolati dalla regione epicardico miocardico e stimolati con 48/80 composto (10μg/mL) e sostanza P (10μM). <img alt="Figura 4" src="/files/ftp_upload/3020/3020fig4.jpg" /> Figura 4. Rappresentante trame citometria a flusso dispersione dei CD4 + isolate cellule marcate con sostanza P (1:100 dilutione). <img alt="Figura 5" src="/files/ftp_upload/3020/3020fig5.jpg" /> Figura 5. Citometria a flusso rappresentante di isolate cellule CD8 + etichettati con Sostanza P (diluizione 1:100).

<ol>
	<li>Brower, G. L., Chancey, A. L., Thanigaraj, S., Matsubara, B. B., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+effect+relationship+between+myocardial+mast+cell+number+and+matrix+metalloproteinase+activity.">Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 283, H518-H525 (2002).</li><li>Brower, G. L., Janicki, J. 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D., Holland, M., Hauer-Jensen, M., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+adverse+myocardial+remodeling+secondary+to+chronic+volume+overload+in+mast+cell+deficient+rats.">Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats.</a> Journal of Molecular and Cellular Cardiology. 43, 56-61 (2008).</li><li>Levick, S. P., McLarty, J. L., Murray, D. B., Freeman, R. M., Carver, W. E., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Mast+Cells+Mediate+Left+Ventricular+Fibrosis+in+the+Hypertensive+Rat.">Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat.</a> Hypertension. 53, 1041-1047 (2009).</li><li>Batista, M. L., Santos, R. V., Cunha, L. M., Mattos, K., Oliveira, E. M., Seelander, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+pro-inflammatory+cytokine+production+and+peritoneal+macrophage+function+in+rats+with+chronic+heart+failure.">Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure.</a> Cytokine. 34, 284-290 (2006).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+Cells:+Versatile+regulator+of+inflammation,+tissue+remodeling,+host+defense+and+homeostasis.">Mast Cells: Versatile regulator of inflammation, tissue remodeling, host defense and homeostasis.</a> Journal of Dermatological Science. 49, 7-19 (2008).</li><li>Yndestad, A., Holm, A. M., Muller, F., Simonsen, S., Froland, S. S., Gullested, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+inflammatory+cytokines+and+activation+markers+in+T-cells+from+patients+with+chronic+heart+failure.">Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure.</a> Cardiovasc. Res. 60, 141-146 (2003).</li><li>Bozkurt, B., Kribbs, S. B., Clubb, F. J., Michael, L. H., Didenko, V. V., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+Concentrations+of+Tumor+Necrosis+Factor-&#x0251;+Promote+Progressive+Left+Ventricular+Dysfunction+and+Remodeling+in+Rats.">Pathophysiologically Relevant Concentrations of Tumor Necrosis Factor-&#x0251; Promote Progressive Left Ventricular Dysfunction and Remodeling in Rats.</a> Circulation. 97, 1382-1391 (1998).</li><li>Frangogiannis, N. G., Lindsey, M. L., Michael, L. H., Youker, K. A., Bressler, R. B., Mendoza, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, H1462-H1468 (2009).</li><li>Forman, M. F., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+histamine+secretion+during+isolation+of+rat+cardiac+mast+cells.">Spontaneous histamine secretion during isolation of rat cardiac mast cells.</a> Inflammation Research. 53, 453-457 (2004).</li><li>Levick, S. P., Murray, D. B., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+nervous+system+modulation+of+inflammation+and+remodeling+in+the+hypertensive+heart.">Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart.</a> Hypertension. 55, 270-276 (2010).</li><li>Morgan, L., Levick, S., Voloshenyuk, T., Murray, D., Forman, M., Brower, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+isolating+functional+mast+cells+from+the+heart.">A novel technique for isolating functional mast cells from the heart.</a> Inflammation Research. 57, 241-246 (2008).</li></ol>

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Questo metodo di isolare le cellule immunitarie cardiaca permette l&#39;analisi della loro funzione, fenotipo, e la maturità senza l&#39;attivazione indesiderata che si verifica in genere nel corso di una tecnica tradizionale isolamento digestione enzimatica. Il numero medio totale di cellule ottenute con questa tecnica è di circa 400.000 per il cuore di ratto, e colorazione differenziale e l&#39;analisi di citometria a flusso di queste cellule indica popolazione mista di linfociti T (~ 70%), i macrofagi (~ 12%) e mas...

<table border="1"><tbody><tr><td> Nome del reagente e </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Hanks Balanced Salt &#39; </td><td> Sigma </td><td> H4891 </td><td> Una bottiglia fa 1 L di Salt Balance Hanks &#39; Soluzione. Equilibrio ricostituire Hanks &#39;Salt Soluzione come parte della procedura. </td></tr><tr><td> Antibiotico-Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3784 </td><td></td></tr><tr><td> Albumina di siero bovino </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> Flacone da 250 ml Nalogene Filtro </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td> Fisher Scientific </td><td> CAS 7647-01-0 </td><td> Per 6 N, aggiungere volumi uguali di HCL al deionizzataacqua. </td></tr><tr><td> 10 N NaOH </td><td> Fisher Scientific </td><td> SC267 </td><td> Per 5 N, aggiungere volumi uguali di NaOH in acqua deionizzata. </td></tr><tr><td> Doppia acqua distillata </td><td></td><td></td><td></td></tr></tbody></table> Tabella di attrezzature specifiche: <table border="1"><tbody><tr><td> Nome del reagente e </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> 3-O sutura in seta intrecciata </td><td> Ethicon Products </td><td> SA64H </td><td></td></tr><tr><td> Ventilatore piccoli animali </td><td> CWE Incorporated </td><td> SAR-830 </td><td> Impostare la velocità di respirazione a 40,5 respiri al minuto. </td></tr><tr><td> Smusso 18 gauge </td><td> BD Biosciences </td><td> 305199 </td><td> Trim aghi di 1 / 2 a 3 / 4 della sua lunghezza originale per l&#39;uso come un tubo tracheale. </td></tr><tr><td> Teflon Tip (con ago rimosso) di 24-gauge Baxter rapido cath catetere </td><td> Baxter </td><td> 2N111 </td><td> Ridurre la lunghezza punta in Teflon da 1 / 2 a 3 / 4. </td></tr><tr><td> 10 cc siringa </td><td> Fisher Scientific </td><td> 14-823-16B </td><td></td></tr><tr><td> 15 ml Falcon Tubi </td><td> BD Biosciences </td><td> 352097 </td><td></td></tr><tr><td> Sorvall RT6000B centrifuga refrigerata </td><td> Fisher Scientific </td><td> 75-004-377 </td><td> Ricordarsi di pre-raffreddare e conservare a 4 ° C. </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Emocitometro </td><td> Hausser scientifico </td><td> HS-LB-1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

Cardiaco cellule immunitarie stanno guadagnando interesse per i ruoli che svolgono nella rimodellamento patologico in molte malattie cardiache 1-5 Queste cellule del sistema immunitario, che includono i mastociti, le cellule T ei macrofagi;. Immagazzinare e rilasciare una varietà di mediatori biologici attivi tra cui citochine e proteasi, quali triptasi. 6-8 Questi mediatori hanno dimostrato di essere giocatori chiave nel metabolismo della matrice extracellulare, attivando metalloproteinasi della matrice o causare accumulo di collagene modulando il fibroblasti cardiaci &#39;funzione. 9-11 Tuttavia, le tecniche disponibili per isolare le cellule immunitarie hanno cardiaca stato problematico perché usano collagenasi batterica per digerire il tessuto miocardico. Questa tecnica causa attivazione delle cellule immunitarie e di conseguenza una perdita di funzione. Per esempio, i mastociti cardiaci diventano significativamente meno sensibili ai composti che causano la degranulazione. 12 Perciò, abbiamo sviluppato una tecnica che permette di tche l&#39;isolamento di cellule cardiache funzionali del sistema immunitario che porterebbe ad una migliore comprensione del ruolo di queste cellule nelle malattie cardiache. 13, 14 Questo metodo richiede una certa familiarità con la posizione anatomica del ratto processo xifoideo, ascella e legamento falciforme, pericardio e del cuore. Questi punti di riferimento sono importanti per aumentare il successo della procedura e per garantire una maggiore resa di cellule immunitarie cardiaco. Queste isolato cellule cardiache immunitarie possono quindi essere utilizzati per la caratterizzazione di funzionalità, fenotipo, la maturità e co-coltura con altri esperimenti cellule cardiache per ottenere una migliore comprensione delle loro interazioni.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

Questo metodo per isolare le cellule immunitarie funzionale dal cuore offre un&#39;alternativa ai metodi convenzionali di digestione collagenasi, che provoca indesiderati attivazione immunitaria delle cellule, con conseguente diminuzione di risposta di queste cellule. Il nostro metodo di isolamento rese funzionali cellule cardiache immunitario evitando problemi legati alla digestione enzimatica.

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 ...

isolation-of-functional-cardiac-immune-cells

Research

JoVE Journal

Immunology and Infection

14.9K Views.  University of South Carolina- School of Medicine. Questo metodo per isolare le cellule immunitarie funzionale dal cuore offre un'alternativa ai metodi convenzionali di digestione collagenasi, che provoca indesiderati attivazione immunitaria delle cellule, con conseguente diminuzione di risposta di queste cellule. Il nostro metodo di isolamento rese funzionali cellule cardiache immunitario evitando problemi legati alla digestione enzimatica.

Video: Isolation of Functional Cardiac Immune Cells

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 ...

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 ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Rilevamento glicogeno in cellule periferiche mononucleate del sangue con Periodic Acid Schiff colorazione

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolamento di cellule dendritiche dal tratto riproduttivo femminile umano per studi fenotipici e funzionali

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Isolamento dei mastociti peritoneo-derivati e loro caratterizzazione funzionale con Ca2 +-imaging e degranulazione saggi

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...