Siamo grati ai pazienti per la loro partecipazione nella raccolta di tessuto per questi esperimenti. Un DoD OCRP ovarica cancro Academy Award (W81XWH-15-0253) e un pilota premio di Colleen Dream Foundation a Anirban K. Mitra supportato questa ricerca.

Il protocollo segue le linee guida del Consiglio normativo istituzionale dell'Università Indiana.
1. cella preparazione
<ol><li>
Isolamento e coltura di cellule mesoteliali primarie umane
	<ol>
<li>Cellule mesoteliali primarie umane isolate (HPMCs) dall'omento umano come descritto in precedenza17,18 e farle crescere in completano crescita medio [dell'Aquila per volta di Dulbecco Medium (DMEM) contenente 10% di siero fetale bovino, 1% penicillina-streptomicina, gli aminoacidi non essenziali di 1% e 1% vitamine] a 37 ° C e 5% CO2. Nota: I HPMCs sono in genere cresciuta fino a 100% confluenza (Figura 1A).</li>
	</ol>
</li>
	<li>
Condizioni di crescita delle cellule tumorali ovariche
	<ol>
<li>Crescere le cellule tumorali ovariche HeyA8 nel mezzo di crescita completa a 37 ° C e 5% CO2. Nota: Uso HeyA8 cellule coltivate a circa 80% confluenza (Figura 1B).</li>
	</ol>
</li>
</ol>2. coltura cellulare
<ol><li>
Set-up cultura prossimale
	<ol>
<li>Uso Transwell inserti per piastre da 6 pozzetti con una membrana di 10 μm di spessore del tessuto-cultura-trattati in policarbonato con 0,4 μm pori (108 pori/cm2) e una zona di crescita di 4,67 cm2 . Se necessario, ottimizzare per diverse misure ridimensionando il numero delle cellule seminate secondo l'area di superficie di crescita. Medium di crescita completa calda, tripsina e tampone fosfato salino (PBS) a 37 ° C prima dell'uso.</li>
	</ol>
</li>
	<li>
Preparare gli inserti
	<ol>
<li>Per le cellule di HeyA8 sulla superficie inferiore della membrana dell'inserto del seme, rimuovere l'inserto dalla confezione usando pinze sterili e posizionarlo invertito in una piastra di coltura sterile cm 15.</li>
		<li>Utilizzare un piatto di 15 cm, come ha la profondità di ospitare l'inserto invertito, o sostituirlo con qualsiasi contenitore sterile appropriato. A seconda dell'esperimento, è necessario inserire il numero desiderato di inserti nel piatto 15 cm.</li>
		<li>Etichetta di conseguenza i tre controlli e tre condizioni sperimentali con un pennarello sul bordo degli inserti.</li>
	</ol>
</li>
	<li>
Preparare le cellule tumorali
	<ol>
<li>Tripsinizzano le cellule di HeyA8 cresciute alla confluenza di 80% in un pallone da2 di 25 cm (~ 2 x 106 cellule), aggiungere 1 mL di tripsina (0,25%) per 40-60 s e neutralizzare la tripsina con 6 mL di medium di crescita completa.</li>
		<li>Centrifugare le cellule a 500 x g a temperatura ambiente (TA) per 3 min, eliminare il supernatante e risospendere il pellet cellulare in 5 mL di terreno di crescita completa. Contare le celle dal vivo utilizzando il metodo di esclusione del colorante blu di trypan.</li>
	</ol>
</li>
	<li>
Semina le cellule tumorali
	<ol>
<li>Sospendere 100.000 cellule/mL di cellule vive HeyA8 nel mezzo di crescita completa. Attentamente seme 80.000 HeyA8 cellule sospese in 800 μL di medium di crescita completa sul fondo dell'inserto (che è ora rivolto verso l'alto, poiché esso è invertito).</li>
		<li>Seme le cellule per formare una forma a cupola, così le cellule rimangono sull'inserto (Figura 1). Iniziare dal centro della membrana e spostare verso l'esterno in cerchi concentrici, mentre lentamente il pipettaggio. Evitare di andare più di 3 mm dal bordo per impedire qualsiasi fuoriuscita del mezzo. Nota: Il numero delle cellule seminate dipenderà il tasso di crescita delle cellule e la durata dell'esperimento. Il numero di cellule HeyA8 seminato per questa cultura prossimale sono stato ottimizzato e le cellule sarà 80 – 90% confluenti alla fine del terzo giorno.</li>
	</ol>
</li>
	<li>
Collegamento delle cellule di cancro
	<ol>
<li>Coprire il piatto di 15cm e spostare con cautela il piatto contenente gli inserti per l'incubatrice di CO2 . È fondamentale per non disturbare la goccia di medium e cellule sull'inserto in questa fase. Lasciare le cellule a 37 ° C per 4 h per consentire le cellule tumorali di allegare all'inserto.</li>
	</ol>
</li>
	<li>
Preparare le cellule mesoteliali
	<ol>
<li>Dopo circa 4 h, tripsinizzano la HPMCs cresciuta alla confluenza del 100% in un pallone da2 cm 75 (~ 4 x 106 cellule) con 2 mL di tripsina (0,25%) per 1 – 2 min e neutralizzare la tripsina con 12 mL di medium di crescita completa.</li>
		<li>Centrifugare le cellule a 500 x g per 3 min a RT, risospendere il pellet cellulare in 10 mL di medium di crescita completa e contare le celle dal vivo utilizzando il metodo di esclusione del colorante blu di trypan.</li>
	</ol>
</li>
	<li>
Semina le cellule mesoteliali
	<ol>
<li>Sospendere 300.000 cellule/mL di HPMCs live nel mezzo di crescita completa. Aggiungere 2,5 mL di mezzi freschi di sviluppo completo in ciascun pozzetto di una piastra a 6 pozzetti. Attentamente e portare il piatto di 15cm contenente gli inserti fuori alla cappa di biosicurezza. Utilizzando pinze sterili, capovolgere l'inserto e posizionarlo nel pozzetto della piastra 6 pozzetti in modo che è in posizione verticale, con le cellule di HeyA8 attaccate alla superficie inferiore immersa nel mezzo di crescita completa (Figura 1).</li>
		<li>HPMCs seme 450.000 sospeso in 1,5 mL di terreno di crescita all'interno degli inserti con le cellule di HeyA8 attaccate alla superficie rivolta verso la parte inferiore del pozzo (Figura 1) o agli inserti senza eventuali cellule di HeyA8 per i controlli HPMC. Aggiungere 1,5 mL di terreno di coltura senza cellule ai controlli HeyA8.</li>
	</ol>
</li>
	<li>
Cresce la cultura prossimale
	<ol>
<li>Lasciate che la cultura prossimale crescere nell'incubatore CO2 a 37 ° C e 5% CO2 per 72 h. Se necessario, le medie possono essere ricostituite come segue.<ol>
<li>Per l'interno dell'inserto, rimuovere µ l 750 e 750 µ l di terreno di coltura fresco completo.</li>
			<li>Per l'esterno dell'inserto (la piastra 6 pozzetti), prelevare 1 mL e aggiungere 1 mL di terreno di coltura fresco completo. 1 mL è stato scelto per la comodità di cambiare il mezzo in un unico passaggio. Se lo si desidera, 1,25 mL può essere rimosso e sostituito invece.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Raccogliendo le cellule
	<ol>
<li>Raccogliere le cellule dopo 72 h della cultura prossimale. Faccia particolare attenzione in questa fase per evitare contaminazioni delle cellule HeyA8 e HPMCs. Trypsinize le cellule, centrifugare li e lisare il pellet invece di fare l'elettrolisi le cellule sulla membrana per evitare una contaminazione incrociata.</li>
	</ol>
</li>
	<li>
Trypsinizing le cellule
	<ol>
<li>Risciacquare entrambi i lati dell'inserto con PBS 1X.</li>
		<li>Trasferire gli inserti per un nuovo pozzo e aggiungere tripsina. Aggiungere 0,5 mL di tripsina all'interno dell'inserto e aggiungere 2 mL di tripsina alla piastra 6 pozzetti per tripsinizzano le cellule tumorali fissate alla superficie inferiore dell'inserto. Incubare la piastra per 2 min a 37 ° C.</li>
	</ol>
</li>
	<li>
Neutralizzare la tripsina
	<ol>
<li>Aggiungere 1 mL di medium di crescita completa all'interno dell'inserto per neutralizzare la tripsina, pipettare su e giù e poi prendere le cellule e trasferirli in una provetta di raccolta contrassegnati. Aggiungere un ulteriore 2 mL di medium di crescita completa per una completa neutralizzazione. Dovrebbe prestare attenzione a non forare la membrana durante il pipettaggio per evitare una contaminazione incrociata delle cellule.</li>
		<li>Per raccogliere le cellule sul fondo dell'inserto, dispensare la tripsina 2 mL che è stato aggiunto nel passaggio 12.2 sulla superficie inferiore 2 – 3 x affinché le cellule cadono nella parte inferiore della piastra 6 pozzetti. Neutralizzare la tripsina sul fondo del piatto con 6 mL di terreno di coltura e trasferire il contenuto della piastra in una provetta di raccolta contrassegnati.</li>
	</ol>
</li>
	<li>
Raccolta e la lisi delle cellule
	<ol>
<li>Centrifugare le cellule a 500 x g per 3 min e aspirare i media. Risospendere e lisare il pellet cellulare con 0,7 mL di fenolo e guanidina tiocianato-basati - reagente di lisi per l'estrazione di RNA. Isolare il RNA usando qualsiasi kit adatto.</li>
	</ol>
</li>
</ol>3. quantitativa Real-Time Polymerase Chain Reaction
Nota: I passaggi seguenti descrivono una reazione a catena della polimerasi quantitativa in tempo reale (qPCR) per studiare i cambiamenti di espressioni del gene come risultato la cultura prossimale.
<ol><li>Preparare cDNA da campioni di RNA per qPCR. Utilizzare qualsiasi adatto della trascrittasi inversa con un buffer corrispondente e casuale primer per assicurare la trascrizione inversa di tutti gli mRNA (Vedi Tabella materiali).</li>
	<li>Valutare i cambiamenti nei livelli di espressione di mRNA di fibronectina (FN1) ed E-caderina (CDH1) e trasformare il fattore di crescita beta 1 (TGFβ1) da qPCR con gliceraldeide 3-fosfato deidrogenasi (GAPDH) come controllo interno. È possibile utilizzare standardizzato analisi di espressione genica disponibili in commercio (Vedi Tabella materiali) o per disegnare primers.</li>
	<li>Confrontare le cellule HeyA8 in coltura prossimale con HPMCs con i controlli di HeyA8 e il HPMCs in coltura prossimale con i controlli HPMC.</li>
</ol>

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La comprensione del meccanismo di paracrine e juxtacrine segnalazione tra cellule è essenziale per lo sviluppo di una migliore conoscenza del tessuto normale omeostasi e malattia condizioni7,8. Maggior parte paracrine studi vengono condotti attraverso la raccolta di segnalazione condizionata medio da una cella tipo e usarlo per trattare l'altro tipo di cellula. Questo metodo ha un vantaggio nella sua intrinseca semplicità. Tuttavia, esso non accuratamente ricap...

Il ruolo di produttivi interazioni reciproche tra cellule tumorali e del microambiente tumorale nella progressione del tumore è stata ben stabilito ed è diventato degli obiettivi principali della ricerca nel cancro biologia1. Casi simili di segnalazione bidirezionale sono cruciali durante la guarigione delle ferite, le risposte immunitarie, angiogenesi, nicchie di cellule staminali e durante lo sviluppo2,3,4,5,6 , 7 , 8. un tema comune a tutti questi processi biologici è che le cellule rispondono in vari modi a segnali extracellulari da loro microambiente che determinano il destino delle cellule, la fisiologia del tessuto e progressione della malattia. Pertanto, la messa a fuoco, diventato sempre più verso lo sviluppo di una migliore comprensione dei meccanismi coinvolti in tali comunicazioni cellula-cellula. Una maggioranza di tali interazioni coinvolgono paracrine o juxtacrine segnalazione tra le cellule. Segnalazione di paracrine coinvolge la secrezione di fattori di segnalazione specifici da una cella che sono percepiti da recettori corrispondenti su un'altra cella nelle vicinanze, innescando una risposta in esso9,10, mentre juxtacrine segnalazione richiede un contatto diretto tra componenti cellulari delle due cellule coinvolte11,12.
Tale segnalazione è una componente fondamentale nell'omeostasi del tessuto, così come nel microambiente tumorale. Le cellule tumorali beneficiano di fattori paracrini e juxtacrine dalle cellule nello stroma del tumore, compreso cancro-collegati di fibroblasti (CAFs), cellule immunitarie e adipociti13,14,15,16. Il paracrine segnalazione può essere mediata da citochine, chemochine, fattori di crescita, ecc., mentre la segnalazione di juxtacrine coinvolge giustapposti ligandi e recettori come tacca di segnalazione, o interazioni tra integrine e delle loro rispettive proteine della matrice extracellulare. Abbiamo dimostrato l'importanza delle interazioni reciproche tra cellule tumorali ovariche e CAFs, progressione e la metastasi del tumore14. Allo stesso modo, le interazioni delle cellule di carcinoma ovarico metastatico con le cellule mesoteliali che copre il sito delle metastasi regolano chiavi microRNA e fattori di trascrizione nelle cellule tumorali che promuovono la colonizzazione metastatica17, 18.
Maggior parte degli studi sulla segnalazione di paracrine implicano l'uso di un raccolto da tipo una cella per trattare il secondo tipo di cella con il medium condizionato. Mentre questo approccio è stato ampiamente utilizzato, non vengono replicate in modo efficace i livelli di alta concentrazione localizzati del fattore secernuto nel microambiente della cellula ricevente. Inoltre non riesce a riprodurre la cinetica del flusso continuo del fattore secreto prodotto da una cellula e ricevuto dalla cella vicina. Paracrino segnalazione è efficace su brevi distanze in quanto i fattori secreti sono alle concentrazioni necessarie solo in prossimità della cella di origine e tendono a diffondere e diluire fuori come la distanza aumenta. Questa alta concentrazione localizzata di fattore secernuto è essenziale per innescare una reazione nella cella del ricevitore. Inoltre, la risposta nelle cellule del destinatario è anche dipenda l'equilibrio dei fattori appena secrete e loro svuotamento continuo attraverso la degradazione, l'associazione e interiorizzazione nella cellule recettive e diffusione dalla cella di origine. Il medium condizionato può essere concentrato sul conto per le più alte concentrazioni localizzate presenti nel microambiente, ma che non può replicare con precisione l'esatta concentrazione. Inoltre, esso non può simulare la cinetica naturale della produzione e lo svuotamento del fattore coinvolto. Per maggiore precisione replicare segnalazione paracrina e separarla da juxtacrine meccanismi di segnalazione, abbiamo messo a punto un metodo novello cultura prossimale, che coinvolge coltivare due tipi cellulari su entrambe le superfici di una membrana porosa. I pori sono abbastanza piccoli per evitare interazioni juxtacrine e ancora permettere lo scambio di fattori secreti alle alte concentrazioni localizzate. In questo modo, questo sistema mantiene la cinetica di produzione e lo svuotamento dei fattori paracrini.

<table>
	<tbody>
		<tr>
			<td>24 mm Transwell permeable support with 0.4 &#181;m Pore Polycarbonate Membrane Insert</td>
			<td>Corning (Costar)</td>
			<td>3412</td>
			<td>&#8226; 10 &#181;m thick translucent polycarbonate membrane 
			&#8226; Treated for optimal cell attachment 
			&#8226; Packaged 6 inserts in a 6 well plate, 4 plates per case 
			&#8226; Membrane must be stained for cell visibility 
			&#8226; Sterilized by gamma radiation</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning (Falcon)</td>
			<td>353046</td>
			<td>Flat Bottom, TC-treated, sterile, with Lid</td>
		</tr>
		<tr>
			<td>15 cm culture dish</td>
			<td>Corning (Falcon)</td>
			<td>353025</td>
			<td>Sterile, TC-treated Cell Culture Dish</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning (Cellgro)</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning (Cellgro)</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning (Cellgro)</td>
			<td>25-020-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin, 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>Biosafety level II cabinet</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>FN1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01549976_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFB1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs00998133_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDH1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01023895_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini RNA isolation Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>43-688-13</td>
			<td></td>
		</tr>
		<tr>
			<td>HeyA8 ovarian cancer cells</td>
			<td>Obtained from Ernst Lengyel Lab, University of Chicago</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946; Neutralizing Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1835-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a proximal culture method to study paracrine signaling between cells

Le interazioni intercellulari svolgono un ruolo importante in molti processi biologici, tra cui la progressione del tumore, le risposte immunitarie, angiogenesi e sviluppo. Paracrino o juxtacrine segnalazione media tali interazioni. L'uso di un medium condizionato e coculture studi sono i metodi più comuni di discriminare tra questi due tipi di interazioni. Tuttavia, l'effetto di localizzate elevate concentrazioni di fattori secreti nel microambiente durante le interazioni paracrine con precisione non è ricapitolata dal medium condizionato e, così, può portare a conclusioni imprecise. Per ovviare a questo problema, abbiamo messo a punto un metodo di cultura prossimale per studiare segnalazione paracrina. I due tipi cellulari sono coltivati su entrambe le superfici di una membrana di policarbonato spessore µm 10 con 0,4 µm pori. I pori consentono lo scambio di fattori secreti e, allo stesso tempo, inibiscono la segnalazione di juxtacrine. Le cellule possono essere raccolte e lisate sull'endpoint per determinare gli effetti della segnalazione di paracrine. Oltre a consentire per gradienti di concentrazione localizzate di fattori secreti, questo metodo è favorevole agli esperimenti che coinvolgono periodi prolungati di cultura, così come l'uso di inibitori. Mentre usiamo questo metodo per studiare le interazioni tra cellule tumorali ovariche e le cellule mesoteliali che incontrano nel sito di metastasi, può essere adattato a qualsiasi due tipi di cellule aderenti per i ricercatori a studiare paracrine segnalazione in vari campi, compreso lo sviluppo, immunologia e microambiente tumorale.

Le cellule di cancro ovarico metastatico ritrovarvi con cellule mesoteliali presso il sito di metastasi all'interno della cavità peritoneale19. Interazioni paracrine e juxtacrine produttive con l'aiuto di cellule mesoteliali nell'indurre risposte adattative nelle cellule del cancro ovarico, che consentono di riuscita metastasi17,18,20,21 </sup...

Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Interazioni cellulari paracrine e juxtacrine gioca un ruolo importante in molti processi biologici, tra cui la progressione del tumore, le risposte immunitarie, angiogenesi e sviluppo. Qui, un metodo di coltura prossimale viene utilizzato per studiare segnalazione paracrina dove le concentrazioni localizzate dei fattori secreti sono mantenute, evitando il diretto contatto cellulare.

Un metodo di coltura prossimale per studiare segnalazione paracrina tra celle

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

a-proximal-culture-method-to-study-paracrine-signaling-between-cells

Research

JoVE Journal

Biology

9.4K Views.  Indiana University School of Medicine. Interazioni cellulari paracrine e juxtacrine gioca un ruolo importante in molti processi biologici, tra cui la progressione del tumore, le risposte immunitarie, angiogenesi e sviluppo. Qui, un metodo di coltura prossimale viene utilizzato per studiare segnalazione paracrina dove le concentrazioni localizzate dei fattori secreti sono mantenute, evitando il diretto contatto cellulare.

Video: A Proximal Culture Method to Study Paracrine Signaling Between Cells

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

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 ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: un metodo per studiare l&#39;architettura tridimensionale dei genomi.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un sistema per la coltura cellule epiteliali del pigmento Iris a studio Rigenerazione Lens Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Visualizzazione dei Vascolare Ca 2 + Segnalazione Innescato dal paracrini Derivato ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Un alto contenuto di flusso di lavoro di imaging per studiare GRB2 Centri di segnalazione di espressione clonazione

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

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 ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

La stimolazione meccanica indotta calcio Wave Propagation in monostrati cellulari: l&#39;esempio di bovini cellule endoteliali corneali

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Un metodo di coltura prossimale per studiare segnalazione paracrina tra celle

Riepilogo

Esplora Altri Video

Capitoli in questo video

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

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

Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

Hi-C: un metodo per studiare l'architettura tridimensionale dei genomi.

Un sistema per la coltura cellule epiteliali del pigmento Iris a studio Rigenerazione Lens Newt

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di Imperata cylindrica

Visualizzazione dei Vascolare Ca^{2 +} Segnalazione Innescato dal paracrini Derivato ROS

Un alto contenuto di flusso di lavoro di imaging per studiare GRB2 Centri di segnalazione di espressione clonazione

A Novel

La stimolazione meccanica indotta calcio Wave Propagation in monostrati cellulari: l'esempio di bovini cellule endoteliali corneali