Il progetto è stato sostenuto da NIH concedere U01HL098180 dal National Heart, Lung, and Blood Institute. Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Heart, Lung, and Blood Institute e il National Institutes of Health

1. Setup Reagenti 1.1 Dissezione e immagini Media L-15 Leibovitz (L15) è completato con FBS (10%) e penicillina-streptomicina (100 unità / ml di penicillina G sodica e 100 pg / ml di streptomicina solfato) è usato sia durante il raccolto e di imaging di campioni trachea. 2. Shallow Walled Cultura della Camera dell&#39;Assemblea La camera utilizzato per contenere trachea tessuto è mostrato in Figura 1. Il pavimento della camera è il fondo di vetro 35 millimetri capsula di Petri. La parte superiore e laterale della camera è generato mettendo un piccolo pezzo di 0,3 mm di spessore fogli silicone sopra il piatto di vetro. Il bordo del foglio è tagliato per adattarsi al piatto fondo rotondo ed il centro è ulteriormente tagliato per formare una camera centrale poco profonda (vedere i punti 2.1-2.3). In cima a questa assemblea, un vetrino di vetro 18 millimetri rotonda è posto per generare un ambiente avente adatto per l&#39;imaging. <ol><li> Coprire completamente il vetrino in vetro mm 18 round con un quadrato di 0,3 mm di spessore rivestimento di silicone. </li><li> Con forbici normali dissezione netta tagliare il telone di silicone in eccesso intorno al bordo del vetrino (risultante in uno strato circolare di fogli di silicone). </li><li> Con un bisturi affilato tagliare e rimuovere una piazza centrale di fogli di silicone (circa 10 mm quadrati) dal centro del vetrino coperto (formando così la parte superiore e sui lati della camera di imaging). </li></ol> 3. Trachea Raccolta e Preparazione <ol><li> Euthanize topi secondo locale la cura degli animali istituzionale e utilizzare comitato e / o linee guida governative e rimuovere trachea in plastica 35 millimetri capsula di Petri standard abbastanza L15 supporto al tessuto sommergere (autori hanno usato topi appena prima della nascita al giorno di gestazione 16.5, fino alla appena nati, neonatale e adulto animali 2). </li><li> Rimuovere non trachea corrispondente tessuto applicata all&#39;esterno dila trachea utilizzando dissezione smussa. </li><li> Rimuovere la laringe con un taglio trasversale appena sotto la cartilagine cricoide usando forbici micro dissezione (Figura 2A, linea 1). </li><li> Rimuovere i rami bronchiali principali destro e sinistro con un taglio trasversale appena sopra la carena usando forbici micro dissezione (Figura 2A, linea 2). </li><li> Premere delicatamente la trachea con una pinza sottile e lavare il lume 2-3 volte con circa 1 ml di balneazione L15 mezzo con un P1000 e P1000 Pipetman punta a cancellare qualsiasi muco e sangue. </li><li> Tagliare un segmento di anello 3-4 di trachea utilizzando un taglio trasversale con micro forbici dissezione (Figura 2B, linea 1). </li><li> Tagliare longitudinalmente attraverso il centro del muscolo trachealis di questo breve segmento trachea usando forbici micro dissezione (Figura 2B, linea 2). </li><li> Tagliare longitudinalmente attraverso il centro degli anelli di cartilagine tracheale con micro forbici dissezione (Figura 2C </strong&gt;, riga), lasciando due sezioni di trachea tessuto adatto per l&#39;imaging (sezione singolo illustrato nella Figura 2D). </li></ol> 4. Cilia Imaging <ol><li> Trasferimento segmenti di tessuto trachea, lato lumen giù, sul centro di un mm con fondo di vetro piatto 35 cultura con 100-200 ml di L-15. </li><li> Per quantificare cilia flusso generato aggiungere una piccola quantità di microsfere fluorescenti 0,20 ìm alla L-15 bagnando il campione di tessuto trachea (~ 1 x 10 11 particelle / ml o 20 microlitri / ml della Fluoresbrite 2,5% sospensione acquosa microsfere). </li><li> Posizionare la camera di imaging preparato precedentemente (vedere Procedura di 1-3 sopra) in cima alla trachea campione silicone lato del foglio in basso, con il campione trachea situato nel centro della camera murato superficiale formata dal taglio finestra nella lastra di silicone (figura 1 ). </li><li> Premere delicatamente sul vetrino per fissare in posizione e rimuovere qualsiasi eccesso di L-15 supporti dai bordi usinga P1000 e P1000 punta Pipetman. </li><li> Montare il fondo di vetro cultura piatto 35 millimetri e il campione trachea su un microscopio invertito con ottica 100X oggettivi e DIC. </li><li> Utilizzando una macchina fotografica ad alta velocità e il software di acquisizione filmato raccoglie i film di ciglia motilità a + 200 fotogrammi al secondo lungo il ciliato epiteli tracheale del raggomitolati bordo del muscolo trachealis (box, e figura 2D Delineato: esempio ancora immagine da filmato registrato) (Movie supplementare 1). </li><li> Utilizzando FITC immagini epifluorescente e una luce CCD basso (Vedi elenco delle attrezzature di cui sopra), raccogliere i film di movimento microsfere fluorescenti sulla superficie degli epiteli tracheale ciliato per consentire successivamente la quantificazione delle ciglia flow generato (Movie integrativa 2). </li></ol> 5. Quantificazione di Cilia Motilità <ol><li> Caricare DIC film imaged nel pacchetto software ImageJ e seguendo la tecnica descritta da Iain Drummond 13 utilizzare lo strumento linea per tracciare una linea di trama che attraversa le ciglia battere. A &quot;reslice&quot; di questa linea genera un immagine tipo chimografo dove il movimento delle ciglia tutta la linea genera un modello di onda. Il numero di pixel tra ogni picco dell&#39;onda è li misurati (un pixel = un fotogramma filmato) da cui è possibile calcolare il numero di battiti al minuto (cioè Hz). </li><li> Per misurare ciglia generato flussi di carico fluorescenti film tallone nel pacchetto software ImageJ, e usare il plugin MTrackJ 14 per tenere traccia manualmente perline fluorescenti sulla superficie della trachea epiteli e lasciare che il software genera velocità di perline e direzionalità per ciascuna sfera rintracciato. </li></ol> Si deve prestare attenzione quando si utilizza perline fluorescenti per quantificare il flusso come la distanza tra le ciglia battere può influenzare fortemente il flusso misurato grandezza. Il movimento del tallone in Supplemental Movie 2 è un buon esempio di questo. In questo esempio beadmovimento è veloce alla superficie delle ciglia battere e riduce notevolmente spento per perline più lontano nei media balneazione. Per controllare per questo, perline tracciati dovrebbero essere raccolti solo ad una distanza fissa sopra la epiteli ciliato in tutti i campioni per permettere una comparazione corretta da fare. Infine, per ottenere dati rappresentativi sul flusso del fluido, sfere dovrebbero essere monitorati a lungo possibile, preferiamo usare tracce tallone che coprono 10 + cellule ciliate per calcolare il flusso del fluido.

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N., 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=Mouse+model+of+heterotaxy+with+single+ventricle+spectrum+of+cardiac+anomalies.">Mouse model of heterotaxy with single ventricle spectrum of cardiac anomalies.</a> Pediatric Research. 63, 9-14 (2008).</li><li>Tan, S. Y., 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=Heterotaxy+and+complex+structural+heart+defects+in+a+mutant+mouse+model+of+primary+ciliary+dyskinesia.">Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia.</a> J. Clin. Invest. 117, 3742-3752 (2007).</li><li>Zhang, Z., 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=Massively+parallel+sequencing+identifies+the+gene+Megf8+with+ENU-induced+mutation+causing+heterotaxy.">Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 3219-3224 (2009).</li><li>Caruso, G., Gelardi, M., Passali, G. C., de Santi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+scraping+in+diagnosing+ciliary+dyskinesia.">Nasal scraping in diagnosing ciliary dyskinesia.</a> Am. J. Rhinol. 21, 702-705 (2007).</li><li>Leigh, M. W., Zariwala, M. A., Knowles, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ciliary+dyskinesia:+improving+the+diagnostic+approach.">Primary ciliary dyskinesia: improving the diagnostic approach.</a> Curr. Opin. Pediatr. 21, 320-325 (2009).</li><li>Delmotte, P., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+beat+frequency+is+maintained+at+a+maximal+rate+in+the+small+airways+of+mouse+lung+slices.">Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices.</a> Am. J. Respir. Cell Mol. Biol. 35, 110-117 (2006).</li><li>Choe, M. M., Tomei, A. A., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+3D+tissue+model+of+the+airway+wall+and+mucosa.">Physiological 3D tissue model of the airway wall and mucosa.</a> Nat. Protoc. 1, 357-362 (2006).</li><li>Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., Randell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Methods Mol. Med. 107, 183-206 (2005).</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, 1315-1321 (2002).</li><li>Thomas, B., Rutman, A., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupted+ciliated+epithelium+shows+slower+ciliary+beat+frequency+and+increased+dyskinesia.">Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia.</a> Eur. Respir. J. 34, 401-404 (2009).</li><li>Drummond, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cilia+in+zebrafish.">Studying cilia in zebrafish.</a> Methods Cell Biol. 93 (08), 197-217 (2009).</li><li>Meijering, E., Dzyubachyk, O., Smal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+cell+and+particle+tracking.">Methods for cell and particle tracking.</a> Methods Enzymol. 504, 183-200 (2012).</li><li>Sisson, J. H., Stoner, J. A., Ammons, B. A., Wyatt, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-digital+image+capture+and+whole-field+analysis+of+ciliary+beat+frequency.">All-digital image capture and whole-field analysis of ciliary beat frequency.</a> J. Microsc. 211, 103-111 (2003).</li><li>Schwabe, G. 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=Primary+ciliary+dyskinesia+associated+with+normal+axoneme+ultrastructure+is+caused+by+DNAH11+mutations.">Primary ciliary dyskinesia associated with normal axoneme ultrastructure is caused by DNAH11 mutations.</a> Hum. Mutat. 29, 289-298 (2008).</li><li>Shah, 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=Loss+of+Bardet-Biedl+syndrome+proteins+alters+the+morphology+and+function+of+motile+cilia+in+airway+epithelia.">Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3380-3385 (2008).</li><li>Clary-Meinesz, C. F., Cosson, J., Huitorel, P., Blaive, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+on+the+ciliary+beat+frequency+of+human+nasal+and+tracheal+ciliated+cells.">Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells.</a> Biology of the Cell / under the auspices of the European Cell Biology Organization. 76, 335-338 (1992).</li></ol>

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Misura della frequenza del battito delle ciglia (CBF) è relativamente facile usando obiettivi del microscopio ad alta potenza e le immagini veloce acquisizione hardware 13,15, e spiega perché le misure CBF sono alla base della maggior parte degli studi indagano clearance mucociliare durante la salute e la malattia. Tuttavia, mentre la misurazione CBF è essenziale per comprendere la clearance mucociliare, la misurazione della CBF solo ignora l&#39;importanza di fondo di entrambi i flussi generati ciliare e ...

Analisi della funzione di cilia motili nella epiteli delle vie aeree è sperimentalmente importanti per la comprensione dei fattori genetici e ambientali che possono influenzare la clearance mucociliare e la salute polmonare 1. La semplice protocollo sviluppato per l&#39;imaging del mouse epiteli delle vie aeree fornisce un metodo efficiente per interrogare vie respiratorie ciglia motilità nei modelli mutanti e knockout topo e richiedono solo competenze di base in topo tracheale dissezione dei tessuti ed ex imaging in vivo di vie ciglia motilità con videomicroscopia alta risoluzione. Questo protocollo è stato istituito e perfezionato nel corso di una schermata di mutagenesi del mouse su larga scala per consentire una rapida valutazione della funzione delle ciglia mobili (cilia frequenza del battito, forma battito delle ciglia, ciglia flusso generato) in mutanti con cardiopatia congenita associata a heterotaxy 2-5. Le attuali tecniche utilizzate per lo studio delle vie respiratorie ciglia motilità possono essere raggruppati in due l&#39;ex vivo tipo acuto o lontermine ger in approcci sperimentali in vitro. Esperimenti acuti includono ex vivo di visualizzazione nasali / vie aeree biopsie spazzola umani 6,7 e analisi di semplici sezioni trasversali 8 vie respiratorie. Gli approcci in vitro utilizzano varie tecniche di coltura cellulare per produrre fogli di differenziata epiteli ciliato, come nelle culture di interfaccia Liquid Air o respiratori sospensione culture 9-11. Tuttavia, queste vie respiratorie epiteli tecniche reciliation richiedono investimenti molto significativo nel tempo e la formazione prima di tutte le cellule epiteliali ciliate utilizzabili sono prodotte per la sperimentazione (4-6 settimane 9,10). Mentre acuta analisi ex vivo delle vie respiratorie epiteliali biopsie pennello sono comunemente utilizzati per gli studi clinici umani, questo metodo non è utilizzabile in studi del mouse a causa di lesioni aggravate tessuto meccanico 12. La tecnica descritta in questo protocollo per l&#39;analisi del moustracheale e vie aeree epiteli non è solo semplice da eseguire, ma non richiede particolari doti dissezione né le attrezzature specializzate, oltre a quelli standard per l&#39;imaging da videomicroscopia. Ci sono molti vantaggi a questo semplice protocollo. Prima, come mouse trachea raccolta dei tessuti è rapida e facile da eseguire, esso consente la rapida valutazione della funzione cilia vie aeree in un gran numero di topi. Questo può includere analisi acuta degli effetti a breve termine diversi trattamenti in vitro. Secondo, essendo una tecnica ex vivo, l&#39;epitelio ciliato vie respiratorie rimane attaccata ad esso sottostanti tessuti di sostegno e conservano quindi associate percorsi di segnalazione cellulare. Quindi in confronto a in vitro reciliated epiteli delle vie aeree, questa preparazione è una migliore rappresentazione del naturale in ambiente tessuto vivo. Terzo, questo protocollo permette l&#39;acquisizione di una serie di parametri quantitativi differenti che possono fornire la valutazione oggettiva motile cilia funzione. Infine, a differenza di altri metodi attuali per via aerea visualizzazione cilia, questo protocollo permette la visualizzazione delle ciglia perpendicolarmente alla direzione battito ciglia, permettendo vista di profilo delle ciglia che è ottimale per l&#39;imaging ad alta risoluzione del battito ciglia e generazione di onda metacronale . Questo protocollo può essere modificato in vari modi per affrontare una vasta gamma di esigenze sperimentali come il ruolo di agenti farmacologici, fattori genetici, esposizioni ambientali, e / o fattori meccanici come carico sulla funzione cilia muco delle vie aeree e la generazione / manutenzione di vie respiratorie battito delle ciglia e propagazione delle onde metacronale.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 Medium </td>
 <td>Invitrogen </td>
 <td>21083-027 </td>
 <td>No phenol red </td>
 </tr>
 <tr>
 <td>Fetal bovine serum </td>
 <td>Hyclone </td>
 <td>SH30088.03 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin </td>
 <td>Invitrogen </td>
 <td>15140-122 </td>
 <td></td>
 </tr>
 <tr>
 <td>2x fine forceps </td>
 <td>Roboz </td>
 <td>RS-4976 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5676 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5620 </td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel </td>
 <td>Roboz </td>
 <td>RS-9801-15 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 pipetman </td>
 <td>Gilson, Inc </td>
 <td>F123602 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 tips </td>
 <td>Molecular BioProducts </td>
 <td>2079E </td>
 <td></td>
 </tr>
 <tr>
 <td>18 mm round glass cover slips </td>
 <td>Fisher Scientific </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic 35 mm culture dishes </td>
 <td>Corning </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Glass bottom 35 mm culture dishes </td>
 <td>Warner Instruments </td>
 <td>W3 64-0758 </td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone sheet 0.012" (0.3 mm) thick </td>
 <td>AAA Acme Rubber Co </td>
 <td>CASS-.012X36-63908 </td>
 <td></td>
 </tr>
 <tr>
 <td>0.20 &mu;m diameter Fluoresbrite YG Carboxylate Microspheres </td>
 <td>Polysciences </td>
 <td>09834-10 </td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope, with 100x oil objective and DIC filters </td>
 <td>Lecia</td>
 <td>DMIRE2 </td>
 <td>Brand is not critical. </td>
 </tr>
 <tr>
 <td>100-watt mercury lamp, epifluorescent FITC excitation/emission filters</td>
 <td>Lecia </td>
 <td></td>
 <td>Brand is not critical.</td>
 </tr>
 <tr>
 <td>Microscope stage Incubator</td>
 <td>Lecia </td>
 <td>11521749 </td>
 <td>Not required if imaging cilia at room temperature</td>
 </tr>
 <tr>
 <td>High-speed camera bright field </td>
 <td>Vision Research </td>
 <td>Phantom v4.2 </td>
 <td>Brand is not critical. Must be faster than 125 fps</td>
 </tr>
 <tr>
 <td>High-speed fluorescent camera </td>
 <td>Hamamatsu </td>
 <td>C9100-12 </td>
 <td>Brand is not critical. Must be faster than 10 fps</td>
 </tr>
 <tr>
 <td>Movie analysis software</td>
 <td>National Institutes of Health</td>
 <td>ImageJ with MtrackJ plugin</td>
 <td></td>
 </tr>
 </tbody>
</table>

ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia

È stata stabilita una tecnica ex vivo per l&#39;imaging del mouse epiteli delle vie respiratorie per l&#39;analisi quantitativa della funzione ciglia motile importante per la comprensione funzione della clearance mucociliare. Appena raccolto il mouse trachea è tagliato longitudinalmente attraverso il muscolo trachealis e montato in una camera murata bassa su un piatto fondo di vetro. Il campione trachea è posizionato lungo il suo asse lungo di approfittare del muscolo trachealis ad arricciarsi longitudinalmente. Questo permette l&#39;imaging di movimento ciliare nella vista profilo lungo l&#39;intera lunghezza tracheale. I video a 200 frame / sec si ottengono utilizzando interferenza differenziale microscopia a contrasto e una fotocamera digitale ad alta velocità per consentire l&#39;analisi quantitativa delle ciglia frequenza del battito ciliare e forma d&#39;onda. Con l&#39;aggiunta di perline fluorescenti durante l&#39;imaging, cilia flusso di fluido generato anche può essere determinato. Il tempo di protocollo si estende a circa 30 minuti, con 5 minuti per la preparazione della camera, 5-10 min per campionemontaggio, e 10-15 minuti per videomicroscopia.

Controllo ciglia delle vie aeree deve essere chiaramente visibile e visto da battere in modo coordinato (Movie Supplemental 1; la riproduzione del filmato è rallentato a tempo reale 15%), con un flusso notevole nella direzione di battito delle ciglia (Movie Supplemental 2; la riproduzione di film è al 100% tempo reale). Quantificazione dei film ciglia dovrebbe produrre risultati simili a quello visto in Figura 3. Raccolta di film DIC alta velocità rende possibile la ...

Watch this Scientific Journal Video about Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia at JoVE.com

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Una tecnica semplice e affidabile per la visualizzazione e la quantificazione delle vie aeree ciglia motilità e ciglia flusso generato usando il mouse trachea è descritto. Questa tecnica può essere modificato per determinare come una vasta gamma di fattori influenza cilia motilità, compresi agenti farmacologici, fattori genetici, esposizioni ambientali, e / o fattori meccanici come carico muco.

Metodo ex vivo per l&#39;imaging ad alta risoluzione di Cilia Motilità in roditori Airway epiteli

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

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Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

20.7K Views.  University of Pittsburgh. Una tecnica semplice e affidabile per la visualizzazione e la quantificazione delle vie aeree ciglia motilità e ciglia flusso generato usando il mouse trachea è descritto. Questa tecnica può essere modificato per determinare come una vasta gamma di fattori influenza cilia motilità, compresi agenti farmacologici, fattori genetici, esposizioni ambientali, e / o fattori meccanici come carico muco.

Video: Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Colture cellulari neuronali da Aplysia per imaging ad alta risoluzione di coni di crescita

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Ricerca

Biologia

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Monitoraggio ad alta risoluzione video di locomozione per adulti in Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Preparazione, Imaging, e la quantificazione di Surface batterica motilità Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Mappatura spazio-temporale di motilità in<em&gt; Ex Vivo</em&gt; Preparazioni di intestini

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

A High Resolution metodo per monitorare l&#39;attivazione fosforilazione-dipendente di IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...