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>

Gli autori non hanno nulla da rivelare.

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 border="0" cellpadding="0" cellspacing="0" width="640">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Autoclud Peristaltic pump</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>26G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>21G*3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>1 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>2.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>10 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>25 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Blunted-edged forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Scissors</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">hemostat</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tissue forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Heparin sodium porcine mucosa (perservative free)</td>
			<td style="width:103px;">Sigma Aldrich</td>
			<td style="width:119px;"></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

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

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

Isolamento di Brain-infiltrazione Leucociti

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Ricerca

Immunologia e infezioni

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolamento e analisi di Brain-sequestrati da leucociti<em&gt; Plasmodium berghei</em&gt; ANKA topi infettati

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergetica e il burst ossidativo: Protocolli per l&#39;isolamento e la valutazione dei leucociti umani e piastrine

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Analizzando gli effetti delle cellule stromali per il reclutamento dei leucociti da portata

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

L&#39;isolamento dei leucociti di Human Interface materno-fetale

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

L&#39;isolamento dei leucociti di tessuti murini all&#39;interfaccia materno-fetale

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Isolamento di leucociti infiltranti da mouse pelle utilizzando enzimatica Digest e Separazione Gradient

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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 ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Due-fotone videomicroscopia Imaging dei leucociti durante la risposta immunitaria nel fegato Lipopolysaccharide-trattati del topo

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

Identificazione dei mediatori del T-cell Receptor segnalazione tramite lo Screening di librerie chimiche inibitore

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

La raccolta selettiva di Marginating-polmonari leucociti

Riepilogo

Esplora Altri Video

Capitoli in questo video

Isolamento di Brain-infiltrazione Leucociti

Isolamento e analisi di Brain-sequestrati da leucociti

Bioenergetica e il burst ossidativo: Protocolli per l'isolamento e la valutazione dei leucociti umani e piastrine

Analizzando gli effetti delle cellule stromali per il reclutamento dei leucociti da portata

L'isolamento dei leucociti di Human Interface materno-fetale

L'isolamento dei leucociti di tessuti murini all'interfaccia materno-fetale

Isolamento di leucociti infiltranti da mouse pelle utilizzando enzimatica Digest e Separazione Gradient

La raccolta selettiva di Marginating-epatiche leucociti

Due-fotone videomicroscopia Imaging dei leucociti durante la risposta immunitaria nel fegato Lipopolysaccharide-trattati del topo

Identificazione dei mediatori del T-cell Receptor segnalazione tramite lo Screening di librerie chimiche inibitore