Gli autori desiderano ringraziare il Sig Tam Po Leung, Mr. Wong Chi Kin e lo staff tecnico della Facoltà di Scienze Workshop, Hong Kong Baptist University, per le loro competenze tecniche e consigli per rendere il prototipo di questo feritore.

1. Introduzione alle parti del feritore (Figura 1) <ol><li> Preparare il supporto del perno fissando i perni ferimento a distanze uguali. Tenere i puntali e regolare l&#39;altezza della punta spostando i perni ferimento regolabili su e giù. </li><li> Montare la guida barra regolabile su diverse marche di piastre di coltura a 96 pozzetti e assicurarsi che l&#39;area ferimento è in una posizione fissa. </li><li> Fissare il perno ferimento regolabile stringendo la vite esagonale. Fissare la guida bar in posizione stringendo i tappi a testa esagonale incassata su entrambe le estremità. </li></ol> 2. Impostare la larghezza del supporto del perno (Figura 2) <ol><li> Allentare i tappi a testa esagonale incassata con la chiave esagonale M5. </li><li> Inserire un numero adeguato di regolazione anelli ad entrambi i lati del supporto del perno in modo che le barre di guida si adattano perfettamente con la larghezza della piastra di coltura a 96 pozzetti. NOTA: Corning e piastre Iwaki richiede 1 paio di grandi anelli e 1 paio di piccoli anelli. Falcon e Nunc plAtes bisogno 1 paio di grandi anelli. </li><li> Serrare la brugola esagonale tappi per fissare la barra di guida. </li></ol> 3. Regolare le barre di guida (Figura 3) <ol><li> Montare i perni ferendo con sterili punte di pipette di plastica da 10 microlitri. </li><li> Allentare i tappi a testa esagonale incassata con la chiave esagonale M5. </li><li> Tenere il feritore e immergere i perni ferimento in una colonna di una piastra di coltura a 96 pozzetti. </li><li> Regolare l&#39;altezza delle barre di guida fino a quando tutti i suggerimenti toccare appena il fondo dei pozzetti. </li><li> Serrare i tappi a brugola esagonale. Assicurarsi che il feritore ora si adatta perfettamente al piastra di coltura e che i perni sono ben posizionate nel mezzo dei pozzi. </li></ol> 4. La calibrazione dei perni <ol><li> Tenere il feritore perpendicolare ad una superficie sterile piatta (cioè Petri) e allentare tutte le viti a testa esagonale con la chiave esagonale M3. </li><li> Toccare il feritore fino a quando tutti i suggerimenti in modo uniforme toccare la superficie sterile piatta. &lt;/ Li&gt; <li> Serrare di nuovo le viti esagonali per bloccare i perni in posizione. </li><li> Controllare la regolarità di ogni punta toccando il feritore delicatamente sulla superficie sterile piatta. </li></ol> 5. Scratching cellulare monostrato (figura 4) <ol><li> Seme umano ombelicale cellule endoteliali vascolari su un 96-pozzetti di coltura cellulare di gelatina rivestite 0,1%. cellule di coltura in media 199 integrate con il 20% di siero di calore-inattivato fetale bovino, 1% di penicillina / streptomicina e 0,09 g / L di eparina. Mantenere le cellule in un incubatore umidificato con 5% di anidride carbonica a 37 ° C durante la notte prima di essere utilizzato. Le cellule devono raggiungere il 100% di confluenza prima ferimento. </li><li> Collocare le punte ferimento alla sinistra (o destra) lato di ciascun pozzetto nella stessa colonna della piastra di coltura. Far scorrere il feritore verso l&#39;altro lato del pozzo; assicurarsi che le punte ferimento tocchino il fondo del pozzo. </li><li> Ripetere la graffiare (Passo 5.2) per tutte le colonne. </li><li> Dopo il ferimento, scarti tegli media in ciascun pozzetto e sostituirlo con terreno contenente composti di test freschi. </li></ol> 6. Acquisizione dati e Image Analysis <ol><li> Immagine tutto bene con la ferita meccanica sul monostrato cellulare utilizzando un microscopio a bassa potenza con fotocamera digitale (es obiettivo 10X) immediatamente dopo ferimento (t = 0 h). </li><li> Aggiungere composti di prova se desiderato e incubare per il tempo desiderato. Immagine tutto bene con l&#39;area della ferita dopo il tempo di incubazione desiderato (t = Dh). </li><li> L&#39;utilizzo di immagini software di analisi come ad esempio ImageJ ( <a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> ), misurare l&#39;area ferito l&#39;immagine utilizzando lo strumento a mano libera. </li><li> Calcolare la percentuale di chiusura della ferita: <img alt="Equazione" src="/files/ftp_upload/55411/55411eq1.jpg" /></li></ol>

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. 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=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. 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=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

Gli autori dichiarano di non avere interessi finanziari concorrenti.

Il nostro feritore cella ha diverse caratteristiche uniche nel risolvere i problemi dei test di migrazione delle cellule tradizionali. Il feritore cellule meccanico a 8 canali è realizzato in acciaio inox di alta qualità (acciaio 304) con una lunga durata di vita che può essere sterilizzato in autoclave. Quasi tutti marchi commerciali di piastre di coltura a 96 pozzetti disponibili sul mercato possono essere utilizzati con questo wounder cellule meccanica causa della barra guida regolabile. Il disegno della barra guida regolabile garantisce anche che la zona graffi è al centro di ogni pozzetto. La ferita in una posizione centrale del pozzo può facilitare la cattura delle immagini utilizzando un microscopio. Le immagini possiedono alcune variazioni mentre taken a tempi diversi, ma non influenzano l&#39;analisi dei dati perché quasi il 90% della superficie e può essere visualizzato da microscopio con un obiettivo a basso ingrandimento (cioè 10X). Così, un&#39;analisi semi-alto rendimento può essere eseguita su piastre a 96 pozzetti di coltura 7, &lt;sup class = &quot;xref&quot;&gt; 8. Tuttavia, questo metodo richiede ancora un gran numero di immagini, che richiedono l&#39;analisi dei dati lungo. Questo può essere risolto se un sistema automatico di acquisizione delle immagini è disponibile. Il design dei singoli pin regolabili del feritore permette anche contatto con la superficie piastra a 96 pozzetti per ottenere ferite identiche, lisci e taglienti sul monostrato cellulare. I perni graffi sono puntali di plastica commerciali, che possono essere sterilizzati e smaltiti dopo ogni singolo graffio, e in grado di fornire grande elasticità a contatto con la superficie bene senza danni meccanici. Questi vantaggi possono ridurre ulteriormente la contaminazione incrociata dei campioni durante l&#39;esperimento e l&#39;influenza sul movimento cellulare attraverso l&#39;area dissodata. In futuro, svilupperemo un perno a molla controllato che può eliminare la fase di regolazione in altezza del perno. La fase più critica di utilizzare questo dispositivo è la forza applicata sul monostrato cellulare durante il graffio. Il feritore devesi terrà perpendicolarmente contro la piastra di coltura, in modo che otto punte per pipette tocchino il fondo del pozzo. Infatti, il feritore fornisce un metodo per eseguire il test migrazione cellulare senza l&#39;uso di apparecchiature costose e complicate. Il protocollo per questo feritore è semplice e lineare. In conclusione, il nostro feritore meccanico a 8 canali è un dispositivo facile da usare per i test di migrazione delle cellule che non richiede particolari competenze. Gli utenti possono eseguire il test migrazione delle cellule in piastre da 96 pozzetti con un numero relativamente elevato di campioni in maniera veloce e semplice 9, 10, 11.

La migrazione cellulare gioca un ruolo importante nei processi cellulari, come ad esempio l&#39;embriogenesi, la neurogenesi, l&#39;angiogenesi, la guarigione delle ferite, delle mucose riparazione, epiteliali-mesenchimali-transizioni in fase di sviluppo normale e la situazione della malattia 1. Si tratta di un processo complicato, che richiede un coordinamento delle numerose manifestazioni inter- e intra-cellulari, compresi segnalazione interazioni molecola, la polarizzazione delle cellule, la riorganizzazione del citoscheletro, rimodellamento della matrice, protrusione della membrana e dinamica cellula-cellula adesione modulazione 2. Studiando la migrazione delle cellule, la scoperta e la convalida delle sostanze chimiche o biomolecole che conducono al movimento delle cellule e le relative vie biochimiche possono essere determinati. Questo processo fondamentale può essere utilizzato vantaggiosamente come proxy per varie applicazioni, come il trattamento di malattia e drug targeting. Il saggio ferendo è uno dei saggi di migrazione cellulare 3. Qui, unsingolo campione di cellule viene parzialmente rimosso dai mezzi meccanici per produrre una zona denudata cui le cellule migreranno per coprire l&#39;area. La percentuale di recupero in un particolare momento sarà monitorato. Quando si maneggiano gran numero di campioni, questioni come il costo sperimentale e la contaminazione incrociata all&#39;interno di campioni possono essere problematico. Anche se molti strumenti commerciali e saggi sono disponibili per lo studio migrazione delle cellule 4, 5, 6, molti di loro richiesto attrezzature costose e sofisticate e miglioramenti sono ancora necessari. Per queste ragioni, un wounder cellule meccanico 8 canali (Figura 1) è sviluppato. Il nostro wounder cellule meccanica 8 canali ha incorporato diverse caratteristiche uniche nel risolvere i suddetti problemi. Esso fornisce la flessibilità necessaria per eseguire la migrazione delle cellule / ferendo saggi nel formato piastra di coltura a 96 pozzetti. La regolazione guprogettazione bar Iding assicura che la zona zero è in posizione centrale di ogni bene. Inoltre, l&#39;altezza delle barre di guida può essere regolato in modo che il feritore è applicabile per varie marche di piastre di coltura. Infine, il design pin ferimento regolabile consente un ancora di contatto delle punte delle pipette con la superficie della piastra di coltura per ottenere simultaneamente e riproducibile ferimento 7, 8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>Nunc</td>
			<td>167008</td>
			<td>Other brands of 96-well cell culture plate can also be used</td>
		</tr>
		<tr>
			<td>P10 pipette tips</td>
			<td>Axygen</td>
			<td>301-03-051</td>
			<td>Short P10 pipette tip is more easy to create a clear wound</td>
		</tr>
		<tr>
			<td>Wounder</td>
			<td>R&#38;P Technology Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M2520-1L</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
	</tbody>
</table>

a device for performing cell migration/wound healing in a 96-well plate

La migrazione delle cellule / ferendo test è un metodo comunemente utilizzato per studiare la migrazione delle cellule e altri processi biologici, come l&#39;angiogenesi e metastasi tumorali. In questo saggio, le cellule sono coltivate per formare un monostrato confluente e una ferita meccanica viene creato graffiando con un dispositivo. Allora il tasso di migrazione delle cellule verso la zona denudato può essere monitorato mediante imaging. Il nostro wounder meccanica 8 canali è progettato per affrontare la maggior parte dei problemi associati con il test migrazione cellulare. In primo luogo, la nostra wounder può essere facilmente sterilizzato in autoclave o con comuni disinfettanti. In secondo luogo, i singoli pin regolabili permettono anche contatto con la piastra di coltura cellulare in modo che possono essere creati ferite taglienti e riproducibili. In terzo luogo, le barre di guida su entrambi i lati del wounder assicurano posizione ferimento consistente in ciascun pozzetto. L&#39;uso di puntali di plastica per il ferimento può fornire ulteriori migliore gestione del feritore e minimizzare le contaminationico. In conclusione, il nostro wounder cella può fornire ai ricercatori un dispositivo facile e riproducibile utente per l&#39;esecuzione del test migrazione cellulare utilizzando la piastra standard coltura a 96 pozzetti.

Questo wounder meccanico 8 canali è costruito graffiare un monostrato di cellule al fine di eseguire il saggio migrazione cellulare. Si tratta di un dispositivo di facile utilizzo in grado di svolgere cellulari graffiare test in piastre da 96 pozzetti in meno di un minuto senza formazione specifica. Questo wounder può introdurre aree avvolte su monostrati di cellule con una larghezza uniforme di circa 600 micron e con spigoli vivi (figure 5 e 6). La larghezza delle ferite dipende dalla marca di puntali e prestazioni dell&#39;utente. Questa gamma di larghezza ferita è adatta per un&#39;immagine comune catturare sotto ingrandimento bassa potenza (Figura 7). Dopo aver catturato l&#39;immagine, la percentuale di chiusura della ferita può essere calcolato. Questo feritore cella ha incorporato diverse strategie per risolvere i problemi riscontrati con il test migrazione delle cellule. Inoltre, esso fornisce flessibilità nella saggi di migrazione cellulare in vari 96 pozzetti cultura plate formati. <img alt="Figura 1" src="/files/ftp_upload/55411/55411fig1.jpg" /> Figura 1: otto canali feritore cellule meccanica. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/55411/55411fig2.jpg" /> Figura 2: Regolare la larghezza del supporto del perno. Inserire gli anelli di regolazione in modo da adattarsi alla piastra a 96 pozzetti. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/55411/55411fig3.jpg" /> Figura 3. Regolare le barre di guida per adattarsi diversi formati lastra. </strong&gt; Far scorrere la barra di guida su e giù per adattarsi alla piastra a 96 pozzetti. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/55411/55411fig4.jpg" /> Figura 4: Graffio direzione della punta. Graffiare le celle trascinando il wounder da un lato del fondo del pozzetto verso l&#39;altro lato del fondo del pozzetto. <img alt="Figura 5" src="/files/ftp_upload/55411/55411fig5.jpg" /> Figura 5: Panoramica della piastra a 96 pozzetti dopo il ferimento. Il monostrato cellulare è ferito dal feritore producendo bordi taglienti con larghezza uniforme di circa 600 micron. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> &lt;<html> / P&gt; <img alt="Figura 6" src="/files/ftp_upload/55411/55411fig6.jpg" /> Figura 6: Immagini rappresentative di una riga ed una colonna di pozzi subito dopo graffi. L&#39;analisi quantitativa della larghezza della ciascuna ferita. Le immagini vengono catturate con un obiettivo 10X. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig6large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 7" src="/files/ftp_upload/55411/55411fig7.jpg" /> Figura 7: catturato le immagini della ferita (obiettivo 10X) a T = 0 e T = h Dh. La linea tratteggiata indica l&#39;area della ferita e la linea continua indica la zona migrazione cellulare. A) vena ombelicale cellule endoteliali umane. B) fibroblasti del derma umano.ottenere = &quot;_ blank&quot;&gt; Clicca qui per vedere una versione più grande di questa figura.

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Qui, presentiamo un protocollo per eseguire un test di migrazione cellulare semi-high throughput su una piastra di coltura cellulare a 96 pozzetti. Questo protocollo è un metodo rapido, semplice ed economico per creare ferite scratch coerenti su un monostrato di cellule.

Un dispositivo per realizzare la migrazione delle cellule / Wound Healing in una piastra a 96 pozzetti

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

a-device-for-performing-cell-migrationwound-healing-in-a-96-well-plate

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.4K Views.  Hong Kong Baptist University.  Qui, presentiamo un protocollo per eseguire un test di migrazione cellulare semi-high throughput su una piastra di coltura cellulare a 96 pozzetti. Questo protocollo è un metodo rapido, semplice ed economico per creare ferite scratch coerenti su un monostrato di cellule. 

Video: A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Capire in anticipo Organogenesis Utilizzando una semplificata<em&gt; In Situ</em&gt; Protocollo di ibridazione in<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

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Biologia dello sviluppo

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Automated quantificazione di cellule ematopoietiche - Interazioni cellule stromali in istologici Immagini di Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

Scalabile piastra a 96 pozzetti Based iPSC cultura e di produzione utilizzando un sistema robotico Liquid Handling

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Istituzione di un clinicamente rilevanti<em&gt; Ex Vivo</em&gt; Mock chirurgia della cataratta modello per indagare epiteliale Wound Repair in un microambiente Native

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Microiniezione per Transgenesi e Genome Editing in Threespine spinarelli

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Adenovirale Terapia genica per diabetici cheratopatia: Effetti sulla guarigione delle ferite e Stem Cell Marker Expression in Human Organ cornee-coltura e le cellule epiteliali Limbal

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Isolamento rapido di BMPR-IB + derivate da tessuto adiposo cellule stromali per l&#39;uso in un cranica difetto di guarigione Modello

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Una tecnica novel di trapianto di grassi del grasso mammario per visualizzare la generazione di vascelli delle cellule staminali endoteliali vascolari

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Generazione di insulina senza impalcatura, tridimensionale esprimendo Pancreatoids dal Mouse progenitori pancreatico In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizzare il nodo e la piastra Notochordal In embrioni di topo Gastrulating mediante microscopia elettronica a scansione e intero Monte immunofluorescenza

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...