Vorremmo riconoscere gli indirizzi di massima Stem Cell FACS centro di ricerca e in particolare ringrazio Jessica Scholes e Felicia Codrea per il loro aiuto con la selezione delle cellule. Il lavoro è stato finanziato dal CIRM RN2-00904-1, K08 HL074229, American Thoracic Society / BPCO Fondazione ATS-06-065, la Fondazione preoccupazione, Il toracica UCLA Jonsson Comprehensive Cancer Center Oncology Program / Lung Cancer SPORE, l&#39;Università della California Cancer La ricerca e il Comitato di Coordinamento Gwynne Hazen Cherry Memorial Laboratories (BG).

Schema delle fasi di 1. Dissezione della trachea 2. Pulizia trachea e tagliarlo 3. Enzima digestione e la trasformazione in sospensione singola cella 4. La colorazione per FACS e l&#39;ordinamento 5. Lavorazione di cellule filtrate per in vivo e in vitro 1. Dissezione di Trachea <ol><li> Euthanize il mouse con una iniezione intraperitoneale di 0,1 cc mg/0.2 di pentobarbital. </li><li> Tagliare la parete addominale, e spostare l&#39;intestino verso il lato per esporre l&#39;aorta addominale e poi sezionarlo. </li><li> Aprite la cassa, tagliando attraverso il diaframma e della parete toracica anteriore su entrambi i lati per esporre il cuore ei polmoni. Continua tagliando nel collo rimuovendo le ghiandole salivari e grassi per esporre la trachea. </li><li> Togliere il timo e tagliare il polmone al largo delle sue connessioni sotto e dietro. Separmangiato la trachea dall&#39;esofago inserendo una pinza tra loro poi tagliare la trachea nella sua parte più alta sopra la sua connessione con la laringe. Quindi tenete la trachea con una pinza e sezionarlo dal suo attaccamento e continuare verso il basso per rimuoverlo con il cuore e polmone en-blocco. Tagliare il cuore ei polmoni, vicino alla ilo includere la maggior parte dei bronchi principali destro e sinistro. </li><li> Posizionare la trachea in una piccola scatola di Petri contenente la raccolta media su ghiaccio. </li></ol> 2. Pulizia Trachea e Cutting It <ol><li> Sotto il microscopio da dissezione, utilizzare pinze punto molto fini (n ° 5) e Vannas forbici Tübingen (FST 15003-08 finescience.com) per pulire la trachea da tutti gli altri tessuti ad esso. Ciò include: linfonodi, nervo laringeo ricorrente, grassi, tiroide e le restanti vasi polmonari. La parte superiore della trachea richiede un&#39;attenzione particolare, per rimuovere le cartilagini laringee grandi senza compromettere il più grande gruppo di SMGche è stata presentata tra la cartilagine cricoide e il primo anello tracheale cartilagineo (C1) 4. </li><li> Tagliare la trachea nella parte superiore e la parte inferiore tra C4 e C5 e quindi tagliare aprire entrambe le parti attraverso il lume della trachea. </li></ol> 3. Digestione enzimatica e Trasformazione in sospensione singola cella (SCS) <ol><li> Incubare la parte inferiore della trachea in 1 ml di 16 dispasi U in una provetta Eppendorf a temperatura ambiente (RT) per 30 minuti, come precedentemente descritto 5. Quindi aggiungere 0,5 mg / ml DNasi I in DMEM per ulteriori 20 minuti a RT. </li><li> Rimuovere le trachee digeriti in fresco medio di raccolta in una piastra di Petri sterile, e spellare l&#39;epitelio superficiale sotto il microscopio da dissezione. Utilizzare una pipetta per trasferire il terreno contenente l&#39;epitelio spogliato in una provetta da 50 ml. </li><li> Centrifugare la epitelio strippato a 1000 xga 4 ° C. Rimuovere il surnatante e aggiungere 0,1% tripsina / EDTA e incubare agitando °in un tubo e 37 ° C agitando incubatore (200 rpm) per 30 min. Dopo l&#39;incubazione, utilizzando una punta 1000 microlitri, pipettare su e giù per rompere grumi e formano un&#39;unica sospensione cellulare. </li><li> Posizionare la trachea superiore in un tubo conico contenente 1 ml di 0,15% pronasi. Incubare a 4 ° C per 4 h. </li><li> Vortex delicatamente il tubo e quindi inserire la trachea nel terreno raccolta fresca e confermare sotto il microscopio da dissezione che c&#39;è completo distacco dell&#39;epitelio di superficie (SE). </li><li> Tritate la trachea con una forbice fine per aprire i vani SMG e poi incubare per un&#39;ora di più in pronasi a temperatura ambiente con agitazione lenta. </li><li> Centrifugare il tessuto tracheale, rimuovere la pronasi, aggiungere 0,1% tripsina / EDTA e incubare a 37 ° C per 30 minuti con agitazione come sopra. </li><li> Passage le cellule digeriti, e gli eventuali grumi bei pezzi rimanenti di ghiandole attraverso aghi 20G, 23G e 26G utilizzando una siringa da 10 ml. Ripetere i tempi passaging più con ogni formato dell&#39;ago primadimissioni. Ora, la maggior parte delle cellule dovrebbe essere in una SCS. </li><li> Filtrare la sospensione attraverso un colino 40 pm quindi lavare il filtro e far girare le cellule. Poi ricostituire le cellule nel volume appropriato del mezzo, contare le cellule e procedere immediatamente alla colorazione FACS e l&#39;ordinamento. Non lasciare le celle a 4 ° C o su ghiaccio per diverse ore, perché abbiamo scoperto che questo riguarda la redditività e riduce notevolmente la sfera formando efficienza. </li></ol> 4. La colorazione per FACS e ordinamento <ol><li> Prendere numero sufficiente di cellule per la preparazione dei tubi senza macchia e singola compensazione macchiati quindi macchiare il resto delle cellule con anticorpi primari, Trop2 capra e ratto ITGA6, per 15 minuti a temperatura ambiente. Lavare quindi con PBS o la raccolta media, rimozione del surnatante con molta attenzione e rompere il pellet di cellule. </li><li> Colorare con l&#39;appropriato anticorpo secondario, incubare al buio a temperatura ambiente per 10 minuti, lavare e rimuovere il surnatante e quindi rompere il pellet. </li><li> Sulle FACSARIA, dopo aver regolato la tensione e la compensazione, porta i detriti, cellule molto piccole, doppietti cellulari e cellule morte. Quindi, la maggior parte delle cellule del vento dovrebbe essere TROP2 positivo, mentre il 20-50% delle cellule SMG sarà TROP2 + 6. </li><li> Impostare la porta di ordinamento per Trop2 ITGA6 + + cellule di SE (cellule basali) e per Trop2 + cellule (+ /-ITGA6 cellule positive) da SMG (cellule del dotto). </li></ol> 5. La lavorazione di cellule Ordinati per: coltura in vitro e il modello in vivo 5.1 In ambito vitro la formazione di 5-9 cultura <ol><li> Miscelare 100 ml di &quot;fattore di crescita ridotto Matrigel&quot; con 100 pl di mezzo completo 10,11 (vedi Tabella 1) contenente fino a 50.000 cellule filtrate poi mettere questa miscela nella camera superiore di un 24-ben trans-well. Poi, mettete 400 ml di terreno completo in camera inferiore e incubare a 37 ° C. Variazione media camera inferiore a giorni alterni e di aggiungere ulteriori 100 pl di Matrigelogni settimana o prima, se diventa sottile. </li></ol> 5.2 Nel modello in vivo <ol><li> Ricostituire la cella ordinati pellet in mezzo completo miscelato 1:1 con Matrigel ad una concentrazione di 5.000-10.000 cellule per 50 pl. </li><li> Anestetizzare la C57/BL6 topi riceventi. Somministrare Carprofen (5 mg / kg) subcutaenously come analgesico. Sterilizzare le spalle e la barba poi fare una incisione cutanea longitudinale centrale nella parte superiore della schiena. L&#39;incisione deve essere abbastanza grande per identificare il sito di iniezione grasso pad nonché la bozza che forma dopo l&#39;iniezione. Utilizzare il lato smussato della forbice di allargare sacche tra la pelle e il retro della parete toracica su entrambi i lati per massimizzare l&#39;accesso e la visualizzazione. </li><li> Identificare il bordo mediale della scapola spingendo il mouse verso l&#39;alto arti anteriori e indietro per vedere il movimento scapola. Prelevare 50 ml di sospensione cellulare in una siringa 300 ml insulina BD e iniettare nel cuscinetto adiposo appena mediale alla scapola e lateralmentele vertebre. Si dovrebbe essere in grado di vedere una bozza altrimenti sono troppo profonde. Ripetere lo stesso sull&#39;altro lato quindi clip o punto l&#39;incisione cutanea. Controllare topi e dare Carprofen (5 mg / kg, SC) una volta al giorno per 48 ore. </li><li> Dopo 3 settimane, i topi con l&#39;eutanasia pentobarbatol (0.1 mg/0.2 cc, IP), aprire la pelle come sopra e sezionare il pad latte intero da entrambi i lati con un margine di sicurezza per evitare di perdere qualsiasi struttura del tessuto che potrebbe essersi formato ed esteso intorno. Fissare in PFA ed embed in paraffina 12. Estrema attenzione deve essere impiegato durante la preparazione e il taglio del blocco di non ignorare o trascurare una struttura. La pratica migliore è quella di tagliare 40 pm una volta quindi tagliare un 4 fetta micron, posizionarlo su un vetrino ed esaminare al microscopio per verificare se ci sono delle &quot;strutture epiteliali&quot; in questa parte del blocco o meno. Se non c&#39;è niente visto, quindi tagliare di nuovo e ripetere l&#39;operazione. </li></ol> 6. Risultati rappresentativi Stripping delle trachee topo si tradurrà in una trachea spoglia che è visibile al microscopio ottico come mostrato nella Figura 1A. Figura 1B mostra la vista di un campo chiaro trachea dopo strippaggio con epiteliale ghiandole della sottomucosa stati rilasciati dai tessuti e grappoli d&#39;uva simili. Pronase rimuove l&#39;epitopo ITGA6 e questo è il motivo per cui i TROP2 cellule del dotto non può essere separato in ITGA6 + e - le popolazioni. Tuttavia, dispasi, che conserva l&#39;epitopo ITGA6, digerisce tutto l&#39;epitelio di superficie e le cellule del dotto anche con tempi di incubazione molto brevi, e pertanto non può essere utilizzato per separare condotto da cellule basali 3. La citometria a flusso delle sospensioni di cellule singole dovrebbe mostrare i diagrammi a dispersione in avanti e laterali che sono rappresentati nella Figura 2A. Buona separazione delle cellule del dotto dal resto se le cellule della trachea deve essere visto con l&#39;anticorpo Trop2 in citometria a flusso per la cella attivata Sorti fluorescenteng (FACS) come mostrato nella figura 2B. Sfere dovrebbe essere visibile nella matrigel dopo circa 1 settimana in coltura e sono densi in apparenza come mostrato in Figura 3. L&#39;efficienza di questo processo è dell&#39;ordine di 1-2% e singole cellule sarà ancora presente in matrigel e probabilmente rappresentano cellule dei dotti delle ghiandole della sottomucosa che non hanno la capacità di auto-rinnovamento. Iniezione di cellule staminali condotto nel grasso mouse pad risultati nella formazione della ghiandola sottomucosa strutture simili che sono mostrate Figura 4. Questi sono visti nei blocchi anche senza colorazione H &amp; E. Questi sono sferiche e possiedono un lume centrale. Molte sezioni devono essere effettuate tramite il cuscinetto adiposo al fine di non perdere queste strutture epiteliali. <img alt="Figura 1" src="/files/ftp_upload/3731/3731fig1.jpg" /> Figura 1. Digestione enzimatica di epitelio tracheale. La rimozione delle cellule epiteliali di superficie viene visualizzato con black frecce, la rimozione delle cellule nei dotti delle ghiandole della sottomucosa è mostrato con frecce verdi. Trachee denudato che sono visibili al microscopio ottico sono presenti dopo 30 minuti di digestione dispasi e dopo 4 ore di digestione pronasi. <img alt="Figura 2A" src="/files/ftp_upload/3731/3731fig2A.jpg" /> Figura 2A. I. La citometria a flusso delle sospensioni di cellule singole che mostrano rappresentante forward scatter e diagrammi a dispersione laterali da trachee del mouse dopo superficie epiteliale stripping. ii. Rappresentante trame FACS per TROP2 espressione in cellule del dotto di trachee del mouse. <img alt="Figura 2B" src="/files/ftp_upload/3731/3731fig2B.jpg" /> Figura 2B. I. La citometria a flusso delle sospensioni singola cellula da epitelio superficiale spogliato dei i due terzi inferiori della trachea del mouse mostra forward scatter rappresentante e diagrammi a dispersione laterali. ii. Tipico diagramma di FACS ITGA6 espressione in surfaccia epitelio. iii. Un altro terreno rappresentante FACS di scatter in avanti e laterale da epitelio superficiale che mostra retro gating della popolazione di cellule basali in verde. iv. ITGA6 tipica e TROP2 esprimere popolazione cellulare in verde. <img alt="Figura 4" src="/files/ftp_upload/3731/3731fig3.jpg" /> Figura 3. Naturalmente il tempo di sviluppo di sfere. Sfere A. Duct. Sfere dovrebbe essere visibile nella matrigel dopo circa 1 settimana di cultura e quasi tutti sono densi in apparenza. Singole cellule che non formano sfere sono anche visto. (Scala bar = 50 micron). Sfere B. basali sono più grandi e luminale in apparenza. Singole cellule che non formano sfere sono anche visto. (Scala bar = 50 micron). <img alt="Figura 4" src="/files/ftp_upload/3731/3731fig4.jpg" /> Figura 4. Iniezione di cellule staminali condotto nel grasso mouse pad risultati nella formazione della ghiandola sottomucosa strutture simili. Rappresentativi delle strutturesono riportati nella cuscinetto adiposo e alcuni sono mostrati produrre mucina (azzurro) con l&#39;Alcian blue acido periodico Schiff macchia.

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	<li>Bartlett, J. A., Fischer, A. J., McCray, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+functions+of+the+airway+epithelium.">Innate immune functions of the airway epithelium.</a> Contrib. Microbiol. 15, 147-163 (2008).</li><li>Finkbeiner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pathology+of+tracheobronchial+glands.">Physiology and pathology of tracheobronchial glands.</a> Respir. Physiol. 118, 77-83 (1999).</li><li>Hegab, 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=A+Novel+Stem/Progenitor+Cell+Population+from+Murine+Tracheal+Submucosal+Gland+Ducts+with+Multipotent+Regenerative+Potential.">A Novel Stem/Progenitor Cell Population from Murine Tracheal Submucosal Gland Ducts with Multipotent Regenerative Potential.</a> Stem Cells. , (2011).</li><li>Jeffery, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+features+of+airway+surface+epithelial+cells+and+glands.">Morphologic features of airway surface epithelial cells and glands.</a> Am. Rev. Respir. Dis. 128, S14-S20 (1983).</li><li>Rock, 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=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 12771-12775 (2009).</li><li>Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trop2+identifies+a+subpopulation+of+murine+and+human+prostate+basal+cells+with+stem+cell+characteristics.">Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20882-20887 (2008).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>McQualter, J. L., Yuen, K., Williams, B., Bertoncello, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+an+epithelial+stem/progenitor+cell+hierarchy+in+the+adult+mouse+lung.">Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 1414-1419 (2010).</li><li>Inayama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+growth+and+differentiation+of+clones+of+tracheal+basal+cells.">In vitro and in vivo growth and differentiation of clones of tracheal basal cells.</a> Am. J. Pathol. 134, 539-549 (1989).</li><li>You, Y., Richer, E. J., Huang, T., Brody, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+mouse+tracheal+epithelial+cells:+selection+of+a+proliferative+population.">Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 283, L1315-L1321 (2002).</li><li>Wu, X., Peters-Hall, J. R., Bose, S., Pena, M. T., Rose, 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=Human+Bronchial+Epithelial+Cells+Differentiate+to+3D+Glandular+Acini+on+Basement+Membrane+Matrix.">Human Bronchial Epithelial Cells Differentiate to 3D Glandular Acini on Basement Membrane Matrix.</a> Am. J. Respir. Cell Mol. Biol. , (2010).</li><li>Ooi, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+a+putative+tumor-initiating+progenitor+cell+population+predicts+poor+prognosis+in+smokers+with+non-small+cell+lung+cancer.">Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.</a> Cancer Res. 70, 6639-6648 (2010).</li></ol>

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Questa tecnica di isolare cellule del dotto e basale dalle vie aeree è importante per migliorare la nostra comprensione della riparazione e rigenerazione delle vie aeree e malattie delle vie aeree. Le tecniche qui descritte sono a pochi passi critici. Il primo è il periodo ottimizzato digestione enzimatica. Il secondo è la creazione di una sospensione singola cellula tramite seriale passaging con aghi calibro progressivamente più elevate per impedire ghigliottina cella ma per rompere ammassi cellulari. Il terzo è l...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Completa media 10: DMEM-F-12, 50/50, 1X) </td><td> Mediatech </td><td> 15-090-CV </td></tr><tr><td> Hepes (15 mm) </td><td> Invitrogen </td><td> 15630 </td></tr><tr><td> Bicarbonato di sodio (3,6 mm o 0,03%) </td><td> Invitrogen </td><td> 25080 </td></tr><tr><td> L-glutammina (4 mM) </td><td> Mediatech </td><td> 25-005-Cl </td></tr><tr><td> Penicillina (100 U / ml) </td><td> Mediatech </td><td> 30-001-CI </td></tr><tr><td> Streptomicina (100 mg / m) </td><td> Mediatech </td><td> 30-001-CI </td></tr><tr><td> Amfotericina B (0,25 mg / ml) </td><td> Lonza </td><td> 17-836R </td></tr><tr><td> Ininsulina (10 pg / ml) </td><td> Sigma </td><td> I6634 </td></tr><tr><td> Transferrina (5 mg / ml) </td><td> Sigma </td><td> T1147 </td></tr><tr><td> Tossina colerica (0,1 mcg / ml) </td><td> Sigma </td><td> C8052 </td></tr><tr><td> Epidermal Growth Factor (25 ng / ml) </td><td> BD </td><td> 354001 </td></tr><tr><td> Bovina estratto ipofisario (30 mg / ml) </td><td> Invitrogen </td><td> 13028-014 </td></tr><tr><td> Siero fetale bovino (5%) </td><td> Pescatore </td><td> SH3008803HI </td></tr><tr><td> L&#39;acido retinoico (0,05 mM) </td><td> Sigma </td><td> R2625 </td></tr><tr><td> Fattore di crescita ridotto Matrigel </td><td> BD </td><td> 354230 </td></tr></tbody></table> Tabella 1. Completare i componenti Media. <table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td&gt; <td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Pronase </td><td> Roche </td><td> 10165921001 </td><td> Usato a 0,15%: -O / N a 4 ° C per isolare la digestione totale delle cellule tracheali per la cultura (ALI) -4 Digestione hr 4 ° C per isolare SMG </td></tr><tr><td> Dispasi </td><td> BD Biosciences </td><td> 354235 </td><td> Usato a 16 unità: 30 min a RT </td></tr><tr><td> DNasi I </td><td> Sigma </td><td> DN25 </td><td> Usato a 0,5 mg / ml: 20-30 min a RT </td></tr></tbody></table> Enzimi Tabella 2. Utilizzato per la digestione enzimatica della trachea.

isolation of basal cells and submucosal gland duct cells from mouse trachea

Grandi vie aeree sono direttamente in contatto con l&#39;ambiente e quindi suscettibile al danno da tossine e agenti infettivi che respiro in 1. Le vie aeree di grandi dimensioni richiedono quindi un meccanismo di riparazione efficace per proteggere i nostri corpi. Questo processo di ripristino si verifica a partire da cellule staminali delle vie aeree e isolare queste cellule staminali delle vie aeree è importante per la comprensione dei meccanismi di riparazione e rigenerazione. E &#39;anche importante per comprendere riparazione anormale che può portare a malattie delle vie aeree 2. L&#39;obiettivo di questo metodo è quello di isolare una nuova popolazione di cellule staminali da tracheali dotti delle ghiandole topo sottomucosi e di inserire in queste cellule in vitro e in vivo sistemi modello per identificare i meccanismi di riparazione e rigenerazione delle ghiandole della sottomucosa 3. Questa produzione mostra i metodi che possono essere utilizzati per isolare e saggio le cellule staminali condotto e basale del grande vie aeree 3. Questo ci permetteràstudiare le patologie delle vie aeree, come la fibrosi cistica, asma e malattia polmonare ostruttiva cronica. Attualmente, non esistono metodi per l&#39;isolamento delle cellule della ghiandola sottomucosa condotto e non ci sono modelli in vivo per studiare la rigenerazione delle ghiandole della sottomucosa.

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Qui mostriamo il nostro protocollo per l&#39;isolamento di cellule della ghiandola basali e sottomucoso condotto da trachee del mouse. Abbiamo anche dimostrare il metodo di iniezione di cellule staminali nel cuscinetto di grasso dorsale del mouse per creare un<em&gt; In vivo</emModello&gt; di rigenerazione ghiandola sottomucosa.

Isolamento delle cellule basali e sottomucosa cellule del dotto di Gland mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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13.3K Views.  David Geffen School of Medicine at UCLA. Qui mostriamo il nostro protocollo per l'isolamento di cellule della ghiandola basali e sottomucoso condotto da trachee del mouse. Abbiamo anche dimostrare il metodo di iniezione di cellule staminali nel cuscinetto di grasso dorsale del mouse per creare un In vivo di rigenerazione ghiandola sottomucosa.

Video: Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolamento di cellule T CD4 + da Linfonodi mouse utilizzando Miltenyi Purificazione MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

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Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Isolamento di cellule staminali della retina dalla Eye mouse

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolamento di cellule staminali del mouse ghiandole salivari

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e coltura di cellule dalla Zona nefrogenica del Rene embrionali di topo

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e analisi funzionale dei mitocondri da cellule in coltura e Tissue mouse

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...