Questo lavoro è sostenuto da sovvenzioni dal Canadian Institutes of Health Research (178.918), Fonds de recherche en santé du Québec - Vision Health Network Research.

NOTA: Maschio C57BL / 6 topi (5-7 settimane) sono da Charles River Laboratories International, Inc. (St-Constant, Quebec, Canada.) E manipolati secondo protocolli approvati dalla Sainte-Justine Comitato Animal Care CHU. Espianti di tessuto bronchiale mouse forniscono una buona fonte di cellule epiteliali bronchiali primarie per lo studio degli effetti proapoptotici di LMPS sulle cellule epiteliali. Questo protocollo descrive la generazione in vitro di LMPS, nonché un metodo per la rilevazione di cellule epiteliali apoptotiche su espianti tissutali bronchiali LMPS trattati. Questo protocollo è costituito da 3 sezioni. 1. LMPS produzione e caratterizzazione NOTA: Per prevenire la contaminazione, in modo che tutti i materiali usati in questo esperimento sono sterili o in autoclave. Eseguire tutti i passaggi a RT in una cappa di sicurezza biologica in condizioni sterili, se non diversamente indicato. 1.1) La stimolazione e Collezione dei deputati9 <ol><li> Scongelare una aliquota di 10 milioni di cellule T CEM in un bagno di 37 ° C dell&#39;acqua. Diluire in 10 ml preriscaldata medio ematopoietiche come X-VIVO, in 15 ml provetta sterile e centrifugare a 200 gx 5 min senza siero. Aspirare le cellule surnatante e risospendere in 5 ml di media pre-riscaldato. </li><li> Trasferire le cellule in un pallone di coltura tissutale T75 (per cellule in sospensione) con 15 ml di media ematopoietiche pre-riscaldato come X-VIVO e incubare per 4 giorni in un incubatore umidificato a 37 ° C con 5% di CO 2. </li><li> Dopo 4 giorni, trasferire tutto il terreno di coltura e le cellule in un pallone di coltura tissutale T175 contenente 100 ml di mezzo fresco. Continuare incubando le cellule per circa 72 ore nelle stesse condizioni sono cresciuti fino ad una densità di 2 milioni di cellule / ml. </li><li> Uniformemente dividere le celle tra quattro beute T175 ciascuna contenente 150 ml di mezzo fresco e continuano coltura cellulare fino cellule sono cresciute (circa 48 ore di incubazione) ad una densità di 2 milioni / ml. </li><li&gt; Raccogliere cellule da ciascuna beuta per centrifugazione a 200 xg per 5 min e risospendere 300 x 10 6 cellule in un pallone nuovo T175 contenente 150 ml di mezzo fresco, per mantenere il 2 milioni / ml densità cellulare. </li><li> Aggiungere actinomicina D (disciolto in DMSO a 2 mg / ml) al mezzo ad una concentrazione finale di 0,5 mg / ml e incubare per 24 ore. </li><li> Trasferire tutto il terreno di coltura in 50 ml provette coniche e centrifugare le cellule a 750 xg per 5 min. Trasferire il surnatante in 50 ml provette coniche e centrifugare a 1500 xg per 15 minuti per rimuovere frammenti di cellule di grandi dimensioni. </li><li> Trasferire il surnatante in una bottiglia da 250 ml e ultracentrifuga a 12.000 g per 50 min. Eliminare il surnatante e raccogliere pellet. </li><li> Pellets Wash LMPS-arricchito con 40 ml di PBS sterile in un tubo da 50 ml per centrifugazione a 12.000 xg per 50 min. Ripetere questa operazione due volte. </li><li> Raccogliere l&#39;ultima surnatante lavaggio; sarà usato come controllo del veicolo. Sospendere il pellet LMPS a 1ml di PBS e trasferire in un 1,5 ml provetta sterile. Aliquota e conservare isolati LMPS a -80 ° C (per evitare più cicli senza disgelo). </li></ol> 1.2) Caratterizzazione dei parlamentari su Analisi FACS 4 <ol><li> Preparare 2 campioni di tampone annexin, 1 con e altre senza CaCl 2: Hepes 10 mM, NaCl 140 mm, più o meno 5 mM CaCl 2. </li><li> Filtro tampone annessina e FACS liquido guaina flusso utilizzando un filtro 0,22 micron per rimuovere le particelle. </li><li> Diluire 1 ml di LMPS in 44 ml di tampone annexin con 5 mM CaCl 2 in un tubo FACS. Preparare un altro tubo di 1 ml di LMPS in 44 ml di tampone annexin senza CaCl 2 (controllo negativo). </li><li> Aggiungere 5 ml di annexinV-Cy5 in ciascuna provetta e mescolare bene. Incubare per 15 minuti a RT al buio. Arrestare la reazione diluendo la miscela con 400 ml di FACS liquido guaina flusso in ciascun tubo. </li><li> Aggiungere 10 ml (200.000 perle) di bea conteggio 7 micronsospensione ds come standard interno in ciascuna provetta per ottenere un conteggio assoluto. </li><li> Stabilire porte di dimensioni relative (FSC-H, PMT E00, log di scala) e relativo granularità (SSC-H, PMT 325, scala logaritmica) trama puntino sul citofluorimetro utilizzando perline fluorescenti dimensioni-calibrato di 1 micron (gate 1) e contando perline cancello 7 micron (gate 2). </li><li> Analizzare il campione LMPS on FSC-H / SSC-H plot utilizzando le porte consolidate e FL-4 canali per annexin (PMT 765, scala logaritmica) diagramma bidimensionale, con l&#39;acquisizione di un segnale fino a 20.000 conteggio sfere sono raggiungibili in porta 2. </li><li> Determinare gli eventi annexinV positivi di LMPS in tampone annexin contenente CaCl 2, e quindi sottrarre gli eventi di LMPS nel buffer annexin senza CaCl 2 (controllo negativo). </li><li> Calcolare il numero assoluto di MP basati sulla seguente equazione: <img alt="Equazione 1" fo:content-width="4in" src="/files/ftp_upload/52651/52651eq1.jpg" /></li></ol> 1.3) Determinazione della MP Protein Concentrazione (Bradford Assay) <ol><li> Preparare 5 diluizioni seriali di standard proteine ​​1,25-20 ug / ml. Pipettare 800 ml di ogni soluzione standard e campione in una provetta pulita in duplice copia. Aggiungere 200 ml di Bradford reagente colorante per ogni provetta. Mescolare bene, poi incubare a temperatura ambiente per 5 min. </li><li> Misurare l&#39;assorbanza a 595 nm. Determinare la concentrazione di proteine ​​della LMPS utilizzando la regressione lineare della curva standard. </li></ol> 2. espianti tessuto bronchiale e LMPS Trattamento NOTA: Prestare particolare attenzione all&#39;ambiente di lavoro sterile e asettico preparare le soluzioni ei media utilizzati in seguito esperimenti. Per preparare la completa guarigione medio, aggiungere 1 ml di tessuto di guarigione medio Supplementi con siero (scongelati su ghiaccio) a 100 ml Tissue guarigione medio e mescolare bene. 2.1) Preparazione di bronchiali espianti di tessuto <ol><li> Prima di coltura, graffiare 6 aree di 1 cm 2ciascuno al bordo della superficie di ogni piatto di coltura tissutale 100 mm con una lama di bisturi. Cappotto ogni graffiato 100 millimetri di tessuto piatto cultura con 2 ml di soluzione di rivestimento piatto cultura, e incubare il piatto in un umidificata CO 2 incubatore O / N a 37 ° C. Vacuum aspirare la soluzione surplus e riempire il piatto con 15 ml di tessuto di lavaggio medio. </li><li> Euthanize C57BL / 6 topi (5-7 settimane) da CO 2 inalazione secondo protocolli approvati dal comitato etico per la cura degli animali. </li><li> Asetticamente sezionare tessuto polmonare con bisturi, Dumont super fine pinzetta, e forbici chirurgiche. Rimuovere con attenzione i vasi sanguigni e parenchima. Mettere tessuto polmonare in ghiaccio-freddo di lavaggio del tessuto medio per il trasporto al laboratorio, se applicabile. </li><li> Ulteriori sezionare bronchi sommerso nel lavaggio dei tessuti Medium e separare bronco con un diametro di 1 a 2,5 mm dal tessuto polmonare periferici. Slice tessuti bronchiali in anelli bronchiali ~ 5 millimetri di spessore con un bisturi. </li&gt; <li> Utilizzare un movimento scavare con le pinze sterili microdissecting curve per raccogliere i frammenti bronchiali e metterli sulle zone graffiate dei piatti. </li><li> Rimuovere il tessuto lavaggio medio, e incubare i frammenti a temperatura ambiente per ~ 5 minuti per consentire loro di aderire ai piatti. </li><li> Aggiungere 10 ml di completa guarigione medio per ogni piatto e metterli in atmosfera controllata camera dell&#39;incubatore modulare. Lavare la camera ad alta O 2 miscela di gas (70% O 2, 25% N 2 e 5% CO 2,). Posizionare la camera in un incubatore orbitale da banco e scuoterla a 37 ° C. Agitare la camera per 24 ore a 10 cicli al minuto per consentire al mezzo di fluire intermittente per i frammenti. </li><li> Dopo 24 ore di incubazione, osservare le espianti di tessuto al microscopio ottico a contrasto di fase invertita. Selezionare espianti bronchiali con assoluta, il movimento capelli fini e vivace dell&#39;epitelio bronchiale per il successivo trattamento LMPS. </li></ol>2.2) LMPS Trattamento <ol><li> Preparare il terreno di coltura completo come segue: media integratori crescita disgelo con siero e fibroblasti inibitore sul ghiaccio. Aggiungere 1 ml di supplementi di media crescita con siero e 200 inibitore fibroblasti ml a 100 ml di terreno di coltura; mescolare accuratamente. Riscaldare il terreno di coltura completo a 37 ° C per 10 minuti prima dell&#39;uso. </li><li> Diluire LMPS isolato in una nuova provetta eppendorf sterile con PBS per preparare uno stock LMPS ad una concentrazione di 800 ug / ml. </li><li> Aggiungere 0,5 ml di mezzo completo Growth a ciascun pozzetto di una piastra di coltura tissutale 12 pozzetti. </li><li> Trasferire gli espianti bronchiali selezionati con le pinze microdissecting curve dal protocollo precedente (punto 2.1) in ciascun pozzetto della piastra di coltura tissutale. </li><li> Etichettare la piastra di coltura appropriato per identificare LMPS pozzi di trattamento e pozzetti di controllo. Aggiungere 25 ml LMPS magazzino in ogni trattamento LMPS bene (per una concentrazione finale di 40 mg / ml) e 25 veicoli di controllo microlitri (vedi LMP<html> produzione s) per i pozzetti di controllo. </li><li> Continuare l&#39;incubazione in atmosfera controllata modulare camera di incubazione a 37 ° C agitando delicatamente. </li><li> Dopo 24 ore, lavare espianti 3 volte con PBS e procedere al (fissaggio 4% paraformaldeide [PFA]) passo successivo. </li></ol> 3. Esame istopatologico 3.1) Preparare i Solutions segue prima di procedere alle prossime tappe <ol><li> Preparare tampone PBS 1x miscelando 137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4, 1.76 mM KH 2 PO 4, pH 7.4. </li><li> Per preparare 4% PFA, sciogliere 20 g di PFA in 400 ml di acqua, riscaldata a 60 ° C sotto agitazione; aggiungere qualche goccia di 10 M NaOH per cancellare la soluzione. Successivamente aggiungere PBS 1x e regolare il volume a 500 ml e il pH a 7,4. Filtro e un&#39;aliquota; conservare a -20 ° C. </li><li> Preparare i seguenti reagenti di disidratazione o di reidratazione; 100%, 90%, 70%, 50% etanolo e xilene. </li></ol>e_step &quot;&gt; 3.2) Espianto Fixation e Sezioni di Tessuto Deparaffinizzazione <ol><li> Mettete ogni espianto in una provetta etichettata con 1,5 ml di 4% PFA e incubare O / N a 4 ° C. Sciacquare gli espianti due volte con 1x PBS. </li><li> Disidratare espianti attraverso una serie di alcool (etanolo al 70%: 3 volte 30 minuti ciascuno; il 90% di etanolo: 2 volte 30 minuti ciascuno; 100% di etanolo: 3 volte 30 minuti ciascuno, quindi xilene: 3 volte 20 minuti ciascuno). Eseguire tutti i passaggi a RT in una cappa aspirante. </li><li> Imbed espianti tissutali in paraffina a 58 ° C in un forno. Preparare 5 micron sezioni di tessuto di spessore con un microtomo rotativo. </li><li> Galleggiante sezioni in un bagno di 56 ° C dell&#39;acqua, e poi montare le sezioni su vetrini istologici etichettati. Porre i vetrini in rack di colorazione manuale e secca a 65 ° C per 1 ora. Lasciare raffreddare i vetrini a temperatura ambiente. </li><li> Immergere le rastrelliere in 4 piatti macchia consecutivi contenenti xilene per 10 minuti ciascuno per rimuovere la paraffina. Immergere le rastrelliere in una serie di etanolo per rimuovere xilene: 100%, quindi il 95%, then 80%, poi 70%, poi 50% di etanolo (5 min per ogni passo). Sciacquare le rastrelliere con acqua di rubinetto per 5 minuti per eliminare l&#39;etanolo. </li></ol> 3.3) ematossilina e eosina (H &amp; E) di colorazione <ol><li> Continuare a lavorare con le sezioni di tessuto fissate; la cremagliera in un piatto di colorazione riempito con ematossilina di Mayer per 15 min. Sciacquare il rack con acqua di rubinetto per rimuovere Hematoxylin per 20 min. </li><li> Mettere in acqua distillata per 30 sec.Place in etanolo al 95% per 30 sec. Mettere in Eosina Y piatto soluzione colorante per 1 min. Disidratare attraverso 2 cambi di etanolo al 95%, 100% etanolo e xilene per 2 minuti ciascuna. </li><li> Eseguire un rapido controllo al microscopio per assicurare che l&#39;eccesso eosina viene rimosso. Mettere 2 o 3 gocce di mezzo di montaggio (Fisher SP15-100) su ogni vetrino, quindi coprire con un vetro di copertura. </li></ol> 3.4) In Situ cella della morte di rilevazione: TUNEL Assay <ol><li> Prima di iniziare, preparare proteinasi soluzione di lavoro K: 20 mg / ml aTris 10 mM / HCl, pH 7.4. </li><li> Ripetere i passaggi da 1 a 5 della Sezione 3.2 (fissazione Espianto e tessuti sezione deparaffinizzazione). Sciacquare i vetrini con deionizzata H 2 O. </li><li> Immergere i vetrini con 1x PBS per 10 min. Scolare l&#39;eccesso di PBS. Incubare sezioni di tessuto per 30 minuti a RT con soluzione di lavoro proteinasi K. Sciacquare scivola due volte con 1x PBS. </li><li> Eseguire il saggio TUNEL come descritto nel manuale di istruzioni del kit di rilevamento morte cellulare. Montare con mezzo di montaggio, e applicare il coprioggetto manualmente con vetrino. </li><li> Analizzare i campioni al microscopio ottico. Utilizzare Image Pro 4.5 per analizzare le cellule apoptotiche in colore marrone. </li></ol>

<ol>
	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, 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=Therapeutic+potential+of+plasma+membrane-derived+microparticles.">Therapeutic potential of plasma membrane-derived microparticles.</a> Pharmacol Rep. 61 (1), 49-57 (2009).</li><li>Yang, C., 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=Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways.">Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.</a> Am J Physiol Regul Integr Comp Physiol. 294 (2), 467-476 (2008).</li><li>Yang, C., Gagnon, C., Hou, X., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells.">Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.</a> Cancer Biol Ther. 10 (5), 448-456 (2010).</li><li>Angelillo-Scherrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-derived+microparticles+in+vascular+homeostasis.">Leukocyte-derived microparticles in vascular homeostasis.</a> Circ Res. 110 (2), 356-369 (2012).</li><li>Maeno, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> J Immunol. 178 (12), 8090-8096 (2007).</li><li>Qiu, Q., Xiong, W., Yang, C., Gagnon, C., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte-derived+microparticles+induce+bronchial+epithelial+cells'+pro-inflammatory+cytokine+production+and+apoptosis.">Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.</a> Mol Immunol. 55 (3-4), 220-230 (2013).</li><li>Martin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shed+membrane+particles+from+T+lymphocytes+impair+endothelial+function+and+regulate+endothelial+protein+expression.">Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.</a> Circulation. 109 (13), 1653-1659 (2004).</li><li>Shet, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+blood+contains+tissue+factor-positive+microparticles+derived+from+endothelial+cells+and+monocytes.">Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes.</a> Blood. 102 (7), 2678-2683 (2003).</li><li>Mause, S. F., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+protagonists+of+a+novel+communication+network+for+intercellular+information+exchange.">Microparticles: protagonists of a novel communication network for intercellular information exchange.</a> Circ Res. 107 (9), 1047-1057 (2010).</li><li>Yang, C., 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=Anti-proliferative+and+anti-tumour+effects+of+lymphocyte-derived+microparticles+are+neither+species-+nor+tumour-type+specific.">Anti-proliferative and anti-tumour effects of lymphocyte-derived microparticles are neither species- nor tumour-type specific.</a> J Extracell Vesicles. 3, (2014).</li><li>Soleti, 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=Microparticles+harboring+Sonic+Hedgehog+promote+angiogenesis+through+the+upregulation+of+adhesion+proteins+and+proangiogenic+factors.">Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors.</a> Carcinogenesis. 30 (4), 580-588 (2009).</li></ol>

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Parlamentari sono mediatori attivi intercellulare diafonia e il loro studio è promettente in molte aree della scienza. 11 Lo studio ha presentato un protocollo dettagliato per la generazione in vitro su larga scala di LMPS derivato da una linea di cellule T apoptotico. Questi parlamentari esprimono un vasto repertorio di molecole di linfociti e sono biologicamente implicati nella regolazione dell&#39;omeostasi cellulare e tissutale. Tuttavia, LMPS provenienti da fonti diverse può essere biologicame...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">LMPs production and characterization</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CEM T cells&#160;</td>
			<td style="width:231px;">ATCC&#160;</td>
			<td style="width:124px;">CCL-119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">X-VIVO 15 medium&#160;</td>
			<td style="width:231px;">Cambrex, Walkersville</td>
			<td>04-744Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T75</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1813.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T175</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1812.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Actinomycin&#160;D&#160;</td>
			<td style="width:231px;">Sigma Chemical Co.</td>
			<td style="width:124px;">A9415-2mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PBS</td>
			<td style="width:231px;">Lifetechnologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#181;m filter</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1826.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Annexin-VCy5</td>
			<td style="width:231px;">BD Pharmagen&#160;</td>
			<td>559933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS flow solution</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">342003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fluorescent microbeads (1 um)</td>
			<td style="width:231px;">Molecular Probes&#160;</td>
			<td style="width:124px;">T8880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysterene counting beads (7 um)</td>
			<td style="width:231px;">Bangs laboratories</td>
			<td style="width:124px;">PS06N/6994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene FACS tubes</td>
			<td>Falcon</td>
			<td style="width:124px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1 ml pipet</td>
			<td style="width:231px;">Fisher</td>
			<td style="width:124px;">13-678-11B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">5 ml pipet</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:124px;">357543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">25 ml pipet</td>
			<td style="width:231px;">Ultident</td>
			<td style="width:124px;">DL-357551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1,5 ml conical polypropylene micro tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">72.690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.554.205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.547.205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">50 ml high speed polypropylene copolymer tube</td>
			<td style="width:231px;">Nalgene</td>
			<td style="width:124px;">3119-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">250 ml high speed polypropylene bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">356011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay (Bradford assay)</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay standard II</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 16x100</td>
			<td>VWR</td>
			<td>47729-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 12x75</td>
			<td>Ultident</td>
			<td>170-14100005B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator&#160;</td>
			<td style="width:231px;">Mandel</td>
			<td style="width:124px;">Heracell 150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Low speed centrifuge</td>
			<td style="width:231px;">IEC</td>
			<td style="width:124px;">Centra8R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed centrifuge</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Avanti J8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 250ml bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JLA16.250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 50ml tube</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JA30.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fow cytometry&#160;</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">FACS Calibur</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Spectrophotometer</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Series 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bronchial tissue explants and sections&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice (5-7 weeks old)&#160;&#160;</td>
			<td style="width:231px;">Charles River Laboratories, Inc.&#160;</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Airway PrimaCell&#8482; System:</td>
			<td style="width:231px;">CHI Scientific, Inc.</td>
			<td style="width:124px;">2-82001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Rib-Back Carbon Steel Scalpel Blades</td>
			<td style="width:231px;">Becton Dickinson AcuteCare</td>
			<td style="width:124px;">371310</td>
			<td>#10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">10003-12</td>
			<td>#7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phase-contrast inverted microscope</td>
			<td style="width:231px;">Olympus Optical CO., LTD.&#160;&#160;&#160;</td>
			<td style="width:124px;">CK2</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">high O2 gas mixture&#160;</td>
			<td>VitalAire Canada Inc.</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">modular incubator chamber</td>
			<td style="width:231px;">Billups-Rothenberg Inc.</td>
			<td style="width:124px;">MIC-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ 4000 incubated orbital shaker</td>
			<td style="width:231px;">Barnstead Lab-Line,&#160;</td>
			<td style="width:124px;">SHKA4000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well tissue culture plate</td>
			<td style="width:231px;">Becton Dickinson and Company</td>
			<td style="width:124px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plastic tissue culture dishes (100 mm)</td>
			<td style="width:231px;">Sarstedt, Inc.</td>
			<td style="width:124px;">83.1802</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Surgical scissors</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">14060-09</td>
			<td style="width:171px;">Straight, sharp, 9cm longth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Half-curved Graefe forceps</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">11052-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">humidified CO2 incubator</td>
			<td style="width:231px;">Mandel Scientific Company Inc.&#160;</td>
			<td style="width:124px;">SVH-51023421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Histopathological examination&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formalin formaldehyde</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">T555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethyl alcohol</td>
			<td style="width:231px;">Merck KGaA, Darmstadt</td>
			<td style="width:124px;">EX0278-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;glutaraldehyde&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">G6403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cacodylate</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">31533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope slides</td>
			<td style="width:231px;">VWR Scientific Inc.&#160;</td>
			<td style="width:124px;">48300-025</td>
			<td>25x75mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">X5-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayer&#39;s hematoxylin</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">MHS16</td>
			<td>Funnel with filter paper&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl&#160;</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.&#160;&#160;</td>
			<td style="width:124px;">A144s-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eosin&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT110116</td>
			<td>Funnel with filter paper&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permount&#8482; Mounting Medium</td>
			<td style="width:231px;">Thermo Fisher Scientific Inc.&#160;</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass coverslip</td>
			<td style="width:231px;">surgipath medical industries, Inc.</td>
			<td style="width:124px;">84503</td>
			<td>24&#215;24&#160;#1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TUNEL detection kit</td>
			<td style="width:231px;">In Situ Cell Death Detection, POD</td>
			<td style="width:124px;">11 684 817 910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">oven</td>
			<td style="width:231px;">Despatch Industries Inc.</td>
			<td style="width:124px;">LEB-1-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">rotary Microtome</td>
			<td style="width:231px;">Leica Microsystems Inc.</td>
			<td style="width:124px;">RM2145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">filter paper</td>
			<td style="width:231px;">Whatman International Ltd.</td>
			<td style="width:124px;">1003150</td>
			<td>#3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:231px;">Nikon Imaging Japan Inc.</td>
			<td style="width:124px;">E800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">staining dish complete</td>
			<td style="width:231px;">Wheaton Industries, Inc.</td>
			<td>900200</td>
			<td>including dish, rack, cover</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1.5 ml eppendorf tube</td>
			<td style="width:231px;">Sarstedt Inc.&#160;</td>
			<td style="width:124px;">72.69</td>
			<td>39x10mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital and Reciprocating Water Bath</td>
			<td style="width:231px;">ExpotechUSA</td>
			<td style="width:124px;">ORS200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate buffered saline&#160;&#160;</td>
			<td style="width:231px;">GIBCO</td>
			<td style="width:124px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fume hood</td>
			<td>Nicram RD Service</td>
			<td>3707E</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

L&#39;interesse per i ruoli biologici delle cellule vescicole membrana derivata nella comunicazione cellula-cellula è aumentato negli ultimi anni. Microparticelle (MPS) sono un tale tipo di vescicole, che vanno a diametro da 0,1 micron a 1 micron, e tipicamente capannone dalla membrana plasmatica delle cellule eucariotiche in fase di attivazione o apoptosi. Qui si descrive la generazione di linfociti T di derivazione microparticelle (LMPS) da apoptosi delle cellule T CEM stimolate con actinomicina D. LMPS sono isolati attraverso un processo di centrifugazione differenziale più gradi e caratterizzate con citometria a flusso. Questo protocollo presenta anche un metodo di rilevamento morte cellulare in situ per dimostrare l&#39;effetto della proapoptotico LMPS sulle cellule epiteliali bronchiali derivate da topi primari espianti tissutali bronchiali respiratorie. Metodi qui descritti forniscono una procedura riproducibile per isolare quantità abbondanti di LMPS da linfociti apoptotici in vitro. LMPS derivatoin questo modo può essere utilizzato per valutare le caratteristiche dei vari modelli di malattia, e farmacologia e test tossicologici. Dato che l&#39;epitelio delle vie aeree offre una barriera fisica e funzionale protettivo tra l&#39;ambiente esterno e del tessuto sottostante, uso di espianti tissutali bronchiali piuttosto che linee cellulari epiteliali immortalizzate fornisce un modello efficace per le indagini richiedono tessuti delle vie aeree.

LMPS sono stati caratterizzati con annessina V colorazione 10 da cellule fluorescenza attivate (FACS) analisi e recintato con 1 micron perle in cui il 97% dei parlamentari (≤1 micron) erano annessina-V-Cy5 positivi (Figura 1A e 1B). In genere, circa 2,5 mg di LMPS stati ottenuti seguendo questo protocollo. Espianti tessuto bronchiale da topi C57BL / 6 sono stati sottoposti a trattamento veicolo e LMPS. Analisi istopatologica delle sezioni bronchiali rivelato l&#39;effetto LMPS sulla integri...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Microparticelle di membrana-capannone cellule (MPS) sono vescicole biologici attivi che possono essere isolate e loro effetti fisiopatologici esaminato in vari modelli. Qui si descrive un metodo per la generazione di parlamentari provenienti dai linfociti T (LMPS) e per dimostrare il loro effetto pro-apoptotico sulle cellule epiteliali delle vie aeree.

Generazione di linfocitica microparticelle e rilevamento del loro effetto pro-apoptotico su cellule delle vie aeree epiteliali

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

generation-lymphocytic-microparticles-detection-their-proapoptotic

Research

JoVE Journal

Immunology and Infection

8.9K Views.  University of Montréal.  Microparticelle di membrana-capannone cellule (MPS) sono vescicole biologici attivi che possono essere isolate e loro effetti fisiopatologici esaminato in vari modelli. Qui si descrive un metodo per la generazione di parlamentari provenienti dai linfociti T (LMPS) e per dimostrare il loro effetto pro-apoptotico sulle cellule epiteliali delle vie aeree. 

Video: Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-coltura Modelli di Pseudomonas aeruginosa Biofilm Cresciuto in diretta cellule delle vie aeree umane

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Ricerca

Immunologia e infezioni

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinazione di nastro adesivo a base di campionamento e di fluorescenza In situ</emIbridazione> per il rilevamento rapido di Salmonella Su prodotti freschi

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Applicazione di un mouse legatura Patch Peyer Assay intestinale Loop per valutare assorbimento da parte delle cellule batteriche M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Metodi per quantitativa individuazione di anticorpi indotta da attivazione del complemento sui globuli rossi

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Large-Scale Purificazione di suino o bovino fotorecettore Outer Segmenti per fagocitosi saggi su cellule retiniche dell&#39;epitelio pigmentato

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Fenotipica e analisi funzionale di cellule T regolatorie attivati ​​Isolato dal linfocitica cronica coriomeningite topi infettati da virus

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Campionamento non invasivo del rivestimento mucoso fluido per la quantificazione dei livelli di Immune-mediatore In Vivo delle vie respiratorie superiore

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Isolamento e caratterizzazione delle microparticelle neutrofilo per studi funzionali

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...