Gli autori e questo lavoro sono stati sostenuti da NIH / NCI concessione # R01CA172138 (a SBE).

Le procedure che coinvolgono soggetti animali sono stati approvati dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC) a Tel-Aviv. 1. Protocollo Rat <ol><li> preparativi <ol><li> Disporre 2 farfalla 21 aghi G. Facoltativamente, preparare 2 farfalla 21 aghi smussati G taglio presentando il bordo tagliente dell&#39;ago. </li><li> Sterilizzare strumenti chirurgici: 2 paia di forbici, pinze smussato-taglio, emostatico, forcipe dente-tessuto sterilizzati in autoclave a 121 ° C per almeno 30 minuti su impostazione gravità (a secco). </li><li> Preparare eparinizzato PBS (30 unità / ml) aggiungendo 30 unità di eparina per ml di soluzione di tampone fosfato (PBS 1x). Utilizzare a temperatura ambiente. 35 ml è il volume minimo necessario per animale. </li><li> Streaming eparina PBS nelle linee pompa peristaltica e gli aghi a farfalla per evitare bolle d&#39;aria. </li></ol></li><li> Perfusione dei polmoni e la raccolta di MP-leucociti <ol><li> Eutanasia l&#39;animale da una dose eccessiva di 8% isoflurano. Confermare l&#39;eutanasia dal monitoraggio respirazione cessazione. </li><li> Al momento della cessazione della respirazione, aprire immediatamente le cavità peritoneale e al torace con le forbici sterili e pinze dente-tessuto. Effettuare una incisione addominale mediana lungo l&#39;addome fino al processo xifoideo. </li><li> Sollevare lo sterno con le pinze, con cautela tagliare la gabbia toracica su entrambi i lati, senza pungere qualsiasi organo interno o grandi vasi sanguigni. Bloccare una pinza emostatica sullo sterno, e rostralmente piegare la gabbia toracica per esporre il complesso cardiopolmonare. </li><li> Inserire un ago a farfalla nel ventricolo destro del cuore, ed entro circa un minuto raccogliere il massimo volume di sangue in una siringa (~ 6 ml da 250 g animale). </li><li> Bloccare la vena cava con un emostatico per evitare la perfusione a ritroso del fegato (vedi la discussione). </li><li> Tenere l&#39;ago a farfalla nel ventricolo destro durante la sostituzione della siringa contenente il sangue con il tubo di efflusso della ppompa eristaltic. </li><li> Inserire un secondo ago a farfalla collegato ad una siringa da 5 ml raccolta nel ventricolo sinistro, evitando la penetrazione del setto interventricolare. </li><li> Accendere la pompa peristaltica ad una velocità di circa 5 ml / min e raccogliere delicatamente nella siringa la prima porzione di perfusato che sono contaminati con il sangue. Continuare fino a quando il colore si trasforma perfusato dallo scuro al rosso pallido. (Scartare il perfuste sangue contaminato). </li><li> Senza cessazione della pompa peristaltica, rapidamente sostituire la raccolta siringa da 5 ml con una siringa da 20 ml raccolta e raccogliere 20 ml di perfusato polmone impiegando una maggiore velocità di perfusione (fino a 7 ml / min). Monitorare continuamente il flusso perfusato nella siringa raccolta, evitando la formazione di vuoto. </li><li> Cessare la portata della pompa peristaltica. </li></ol></li><li> I leucociti Estrazione <ol><li> Centrifugare il perfusato per 10 minuti a 400 × g. </li><li> Aspirare il fluido. </li><li>Aggiungere 10 ml di PBS o medie, centrifugare a 400 xg per 10 min, e aspirare il liquido. Ripetere questa operazione due volte. L&#39;uso di PBS o mezzo dipende il conseguente impiego del campione. </li><li> Aggiungere 20 ml di PBS o medie, centrifugare a 400 xg per 10 min, e aspirare il liquido. </li><li> Ricostituire cellule alla concentrazione desiderata, basati sull&#39;utilizzo campione (FACS, PCR, etc.). </li></ol></li></ol> 2. Il protocollo del mouse <ol><li> preparativi <ol><li> Disporre 2 farfalla 25 aghi G. Facoltativamente, preparare 2 farfalla 25 aghi smussati G taglio presentando il bordo tagliente dell&#39;ago. </li><li> Sterilizzare strumenti chirurgici: 2 paia di forbici, pinze smussato-taglio, emostatico, forcipe dente-tessuto sterilizzati in autoclave a 121 ° C per almeno 30 minuti su impostazione gravità (a secco). </li><li> Preparare eparinizzato PBS (30 unità / ml) aggiungendo 30 unità di eparina per ml di soluzione di PBS 1x. Utilizzare a temperatura ambiente. 25 ml è il volume minimo necessario per animale. </li><li> stream eparinizzato PBS nelle linee pompa peristaltica e gli aghi a farfalla per evitare bolle d&#39;aria. </li></ol></li><li> Perfusione dei polmoni e la raccolta di MP-leucociti <ol><li> Eutanasia l&#39;animale da una dose eccessiva di 8% isoflurano. Confermare l&#39;eutanasia dal monitoraggio respirazione cessazione. </li><li> Al momento della cessazione della respirazione, aprire immediatamente le cavità peritoneale e al torace con le forbici sterili e pinze dente-tessuto. Effettuare una incisione addominale mediana lungo l&#39;addome fino al processo xifoideo. </li><li> Sollevare lo sterno con le pinze, con cautela tagliare la gabbia toracica su entrambi i lati, senza pungere qualsiasi organo interno o grandi vasi sanguigni. Bloccare una pinza emostatica sullo sterno, e rostralmente piegare la gabbia toracica per esporre il complesso cardiopolmonare. </li><li> Bloccare la vena cava con un emostatico per evitare la perfusione a ritroso del fegato (vedi la discussione). </li><li> Inserire un ago a farfalla collegato al tubo di efflusso della pompa peristaltica nel ven destratricle del cuore, ed un secondo ago a farfalla collegato ad una raccolta siringa da 2 ml nel ventricolo sinistro del cuore, avoidingpenetration del setto interventricolare. </li><li> Accendere la pompa ad una velocità di circa 2 ml / min e raccogliere i primi millilitri di perfusato sangue contaminato nella siringa di raccolta fino a quando il perfusato si trasforma dallo scuro al chiaro rosso (Scartare il perfuste sangue contaminato). </li><li> Senza cessazione della pompa peristaltica, rapidamente sostituire la raccolta siringa da 2 ml con una siringa da 10 ml raccolta e raccogliere 10 ml di perfusato polmone impiegando una maggiore velocità di perfusione (fino a 4 ml / min). Monitorare continuamente il flusso perfusato nella siringa raccolta, evitando la formazione di vuoto. </li><li> Cessare la portata della pompa peristaltica. </li></ol></li><li> I leucociti Estrazione <ol><li> Centrifugare il perfusato per 10 minuti a 400 × g. </li><li> Aspirare il fluido. </li><li> Aggiungere 10 ml di PBS o mezzo, centrifugare a 400xg per 10 min, e aspirare il liquido. Ripetere questa operazione tre volte. L&#39;uso di PBS o mezzo dipende il conseguente impiego del campione. </li><li> Aggiungere 10 ml di PBS o medie, centrifugare a 400 xg per 10 min, e aspirare il liquido. </li><li> Ricostituire cellule alla concentrazione desiderata, basati sull&#39;utilizzo campione (FACS, PCR, etc.). </li></ol></li></ol>

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, 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+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, 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=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, 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=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, B., 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

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Il metodo qui presentata consente la raccolta selettiva e studiando della popolazione unica di MP-leucociti. Rispetto al circolante o leucociti splenici, la popolazione MP è caratterizzata da una diversa composizione delle sottopopolazioni di leucociti, livelli di attivazione superiori, maggior rilascio di varie citochine, e più elevati livelli di mRNA di pro- e citochine antinfiammatorie 2,3. In particolare, abbiamo dimostrato che le cellule MP-NK sono più citotossico cellule NK circolatori contro varie c...

I leucociti aderenti ai capillari dei polmoni (ad esempio, marginating-polmonare (MP) leucociti) 1 sono stati mostrati ad esporre distinti composizione dei leucociti e l&#39;attività unico rispetto ai leucociti da altri comparti del sistema immunitario (ad esempio, la circolazione, milza, midollo osseo) 2- 4. Ad esempio, mostra MP-leucociti superiore cellule natural killer (NK) citotossicità contro varie cellule tumorali, rispetto al circolanti e milza cellule NK, così come i livelli differenziati RNA messaggero (mRNA) e aumento della secrezione di citochine pro e anti-infiammatori. La composizione delle cellule è anche differenziata da leucociti circolanti come MP-leucociti hanno un rapporto più elevato di immunità innata / adattativa rispetto ai leucociti circolanti (50% contro 30%, rispettivamente). L&#39;obiettivo del metodo qui presentato è quello di consentire la raccolta selettiva di MP-leucociti, per studiare questo compartimento immunitario importante ed unico (popolazione cellulare), e elucIDATE l&#39;impatto delle varie manipolazioni (ad esempio, attivazione immunitaria) su queste cellule specifiche. Per comprendere il significato di questa popolazione unica, è importante notare che il sistema immunitario può controllare cellule tumorali, micrometastasi e malattia residua che circola attraverso funzioni in vivo di immunità cellulo-mediata (CMI). Questa capacità è evidente nonostante il fallimento precedente del sistema immunitario di controllare il tumore primario, e supportato da ampia evidenza in vivo in pazienti oncologici e modelli animali 5. È importante sottolineare che questi risultati sono spesso in contrasto con in vitro ed ex vivo studi che riportano che la maggior parte delle cellule tumorali autologhe sono resistenti alla citotossicità dai leucociti nei campioni di sangue che circola da esseri umani e gli animali (misurata mediante saggi di citotossicità) 6,7. Questa discrepanza può essere attribuita alla presenza in vivo di popolazioni leucocitarie distinte, come il giàmenzionata popolazione leucocitaria MP, e in particolare la sua sottopopolazione di cellule NK attivate 3. Infatti, le cellule tumorali singenici (MADB106), che sono risultati essere resistenti alla circolazione e leucociti splenici, hanno dimostrato di essere lisate dalle cellule MP-NK 3,8. Così, le cellule MADB106 presumibilmente &#39;NK-resistenti &quot;che metastatizzano polmoni di fischer344 (F344) ratti sono controllati da cellule MP-NK, ma non da cellule NK spleniche, comunemente studiato a causa della loro facilità di accesso circolante o. Purificata e attivi MP-leucociti sono inaccessibili attraverso i metodi di raccolta standard dei leucociti dai polmoni, che si basano sulla macinazione tessuto polmonare o degradazione biologica 9. Il nostro approccio ha due grandi vantaggi rispetto agli approcci di lavorazione dei tessuti. Innanzitutto, l&#39;approccio perfusione raccoglie selettivamente MP-leucociti, separandole da altre cellule che provengono dal parenchima polmonare, interstiziale, e compar bronco-alveolaretments. In secondo luogo, la tecnica di perfusione meglio preserva l&#39;integrità e l&#39;ambiente fisiologico MP-leucociti, a differenza della macinazione e trasformazione biologica approcci che danneggiano le cellule, alterano la morfologia e inducono la produzione e il rilascio di vari fattori che modulano l&#39;attività immunitaria e specificamente sopprimono NK citotossicità 10. I polmoni sono un importante organo bersaglio per le metastasi del cancro e per varie malattie infettive. Tutte le cellule maligne circolanti e cellule infettate passano attraverso i capillari polmonari, dove devono deformarsi e interagire con le cellule endoteliali dei capillari e leucociti residenti. In queste condizioni, le cellule circolanti possono essere facilmente bersaglio di MP-leucociti residenti. Sembra quindi biologicamente vantaggioso avere leucociti attivati ​​in questo compartimento immunitario, ed è importante studiare questo unico MP-popolazione in impostazioni biologici, sperimentali e clinici differenti. E &#39;degno di nota che syattivazione stemic immune dai vari risposta modificatori biologici (ad esempio, l&#39;acido polyinosinic-policitidilico (poli I: C) o di tipo C CpG oligonucleotidi (CpG-C ODN)) hanno dimostrato di attivare la MP-leucociti più di leucociti circolanti 3, 8,11.

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Heparin sodium porcine mucosa (perservative free)</td><td>Sigma Aldrich</td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

La mostra MP-vano una diversa composizione dei leucociti sottoinsieme rispetto ai leucociti circolanti. Citometria a flusso di analisi, sottopopolazioni di leucociti sono stati identificati e quantificati per caratterizzare la composizione sia del circolante e leucociti MP. Granulociti e linfociti sono stati identificati sulla base disperde in avanti e laterali. All&#39;interno dei linfociti, NKRP-1 le cellule luminose sono stati identificati come cellule NK, CD3 +,...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

La raccolta selettiva di Marginating-polmonari leucociti

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

5.8K Views.  Tel-Aviv University. Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Video: Selective Harvesting of Marginating-pulmonary Leukocytes

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Raccolta murini macrofagi alveolari e Valutare l&#39;attivazione cellulare indotta da nanoparticelle polianidride

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Ricerca

Bioingegneria

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolamento di cellule polmonari mouse dendritiche

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Immunologia e infezioni

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

La raccolta selettiva di Marginating-epatiche leucociti

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

<meta charset="utf8"></head><body>Distinguere le cellule immunitarie intrapolmonari dalle popolazioni di cellule immunitarie intravascolari: l'approccio intragiugulare

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Distinguere le cellule immunitarie intrapolmonari dalle popolazioni di cellule immunitarie intravascolari: l'approccio intragiugulare

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

Raccolta delle cellule endoteliali dalle punte del palloncino del catetere di Swan-Ganz dopo cateterizzazione del cuore destro

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

Medicina

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Analisi citometrica a flusso per l'identificazione delle cellule immunitarie innate e adattative del polmone murino

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

<meta charset="utf8"></head><body>Isolamento di cellule immunitarie e non immunitarie da polmoni di topo per citometria a flusso

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

<meta charset="utf8"></head><body>Isolamento di popolazioni di cellule mieloidi ed epiteliali vive dal polmone di topo

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

Isolamento di popolazioni di cellule mieloidi ed epiteliali vive dal polmone di topo

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

La raccolta selettiva di Marginating-polmonari leucociti

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Distinguere le cellule immunitarie intrapolmonari dalle popolazioni di cellule immunitarie intravascolari: l'approccio intragiugulare

Distinguere le cellule immunitarie intrapolmonari dalle popolazioni di cellule immunitarie intravascolari: l'approccio intragiugulare

Raccolta delle cellule endoteliali dalle punte del palloncino del catetere di Swan-Ganz dopo cateterizzazione del cuore destro

Analisi citometrica a flusso per l'identificazione delle cellule immunitarie innate e adattative del polmone murino

Isolamento di popolazioni di cellule mieloidi ed epiteliali vive dal polmone di topo

Isolamento di popolazioni di cellule mieloidi ed epiteliali vive dal polmone di topo

Isolamento di popolazioni di cellule mieloidi ed epiteliali vive dal polmone di topo