Questo lavoro è stato sostenuto da sovvenzioni dal St, Fondazione Joseph (AJF, Lji) e American Lung Association, RG-224607-N (Lji).

1. Manutenzione Cell Line <ol><li> Umana immortalizzate BE displastiche di alto grado (CP-B, CP-C, CP-D) e metaplastica (CP-A) linee cellulari mediante trasfezione stabile della telomerasi umana trascrittasi inversa (TERT) proteina 11. </li><li> Mantenere BE linee cellulari in mezzi di cheratinociti, completata con estratto di ipofisi bovina (BPE), fattore di crescita epidermico (EGF), siero bovino fetale 5% (FBS), aminoacidi non essenziali (NEAA), penicillina-streptomicina-neomicina (PSN), e norma condizioni di coltura tissutale (37 ° C, 5% di CO 2, il 98% di umidità). </li></ol> 2. BE cellulare placcatura <ol><li> Preparazione di 12-pozzetti / lamelle. Mettere 18 millimetri vetrini in una piastra di Petri di vetro e autoclave. Trasferimento autoclavato 18 millimetri vetrini coprioggetto in un piatto di coltura tissutale a 12 pozzetti utilizzando una pipetta sterile di vetro collegata ad un tubo a vuoto. Lavare il vetrino con PBS sterile e dei media. </li><li> Trypsinize e contare le cellule essere immortalati. Contare le celle dell&#39;USIng un contatore automatico di cellule o un emocitometro. Diluire le cellule in coltura ad una concentrazione di 20.000-40.000 cellule / ml. </li><li> Mettere 1 ml di cellule in ciascun pozzetto contenente un vetrino per un conteggio di cellule totale di 20.000-40.000 cellule / pozzetto. </li><li> Utilizzare un puntale sterile per spingere delicatamente il vetrino sul fondo del pozzo per assicurare che le cellule attribuiscono al vetrino. </li></ol> Trattamento farmacologico delle cellule BE 3. <ol><li> Diluire farmaco selezionato in (37 ° C), i media caldi di concentrazione desiderata e mescolare bene invertendo il tubo più volte o utilizzando un vortex. Concentrazioni di farmaco determinato in modo empirico. Iniziare il trattamento 24 ore a seguito del seeding iniziale delle cellule essere quello di permettere alle cellule attaccamento al vetrino </li><li> Per confronto e controllo, trattare un vetrino aggiuntivo di cellule sia con 0,1% di veicolo (dimetilsolfossido [DMSO] o l&#39;agente usati per solubilizzare composto) diluito in coltura. Targhetta di identificazione con una penna laboratorio per identificare il farmaco e vehicle campioni trattati. Posizionare cellule in un incubatore di coltura cellulare impostato a 37 ° C, 5% CO 2, 98% di umidità e incubare per punti di tempo desiderati. </li></ol> 4. Immunofluorescenza <ol><li> Fissazione <ol><li> Seguendo tempo di incubazione desiderata del trattamento farmacologico, aspirare il supporto e lavare i coprioggetti rapidamente con 1x tampone fosfato salino (PBS) a temperatura ambiente (RT, 25 ° C). </li><li> Aspirare il PBS e aggiungere 1 ml di PBS contenente 4% PFA [4% PFA / PBS] in ciascun pozzetto ed incubare a temperatura ambiente per 30 min. </li><li> Aspirare il 4% PFA / PBS e lavare il coprioggetto 3x, 5 minuti ciascuno con PBS a temperatura ambiente. Il negozio di lamelle per una settimana in PBS/0.1% PFA a 4 ° C se lo si desidera. </li></ol></li><li> Permeabilizzazione / Blocco Permeabilize cellule PFA fisse per consentire l&#39;anticorpo per accedere ai bersagli intracellulari. Per extracellulare superficie proteine, eseguire tutti i passaggi successivi, senza saponina aggiunto alla soluzione BSA 1x PBS/0.5%. <ol><li> Ridurre sfondo incubando con 50 mM NH 4 Cl diluito in PBS per 10 minuti a RT e lavare con PBS RT volta per 5 min. </li><li> Incubare essere cellule per 30 minuti a 100-300 ml di PBS contenente 0,1% e saponina allo 0,5% albumina di siero bovino (BSA). Diluire saponina da filtro sterilizzato 10% stock, preparato in acqua e conservati a 4 ° C. </li></ol> NOTA: Questo impedisce il legame non specifico degli anticorpi verso proteine ​​cellulari bloccando coprioggetti causa dell&#39;aggiunta di 0,5% BSA. In alternativa, sostituire la BSA con siero di capra diluito al 5% per contribuire a ridurre il legame non specifico degli anticorpi. </li><li> Preparazione della Camera umano <ol><li> Preparare un piatto saggio biologico piazza stratificando il fondo della camera con asciugamani di carta bagnati e mettere uno strato di Parafilm sulla parte superiore. </li><li> Delicatamente trasferire i coprioggetti, celle rivolte verso l&#39;alto, sul Parafilm con pinze e un ago da siringa. </li><li> Segnare il Parafilm con una penna di laboratorio per identificare i vetrini. </li></ol></li><li>Incubazione Anticorpo Primario <ol><li> Diluire l&#39;anticorpo primario in PBS contenente BSA sia 0,5% e 0,1% saponina. </li><li> Tamponare delicatamente la soluzione di saturazione usando un tessuto di laboratorio e aggiungere 100 ml di soluzione di anticorpo primario. Incubare l&#39;anticorpo primario sulle cellule BE notte a 4 ° C. </li></ol></li><li> Incubazione secondario <ol><li> Lavare i vetrini 3x con PBS/0.5% BSA/0.1% saponina per 5 minuti ciascuno a RT. </li><li> Diluire anticorpo secondario coniugato con un tag fluorescente in PBS/0.5% BSA/0.1% saponina che l&#39;anticorpo secondario riconosce le specie in cui l&#39;anticorpo primario è stato sollevato. </li><li> Aggiungere 100 ml di anticorpo diluito ad ogni vetrino. Incubare l&#39;anticorpo secondario per 2 ore a temperatura ambiente. </li></ol></li><li> Lavaggio e Montaggio di Coprivetrini <ol><li> Lavare il coprioggetti 3x, 5 min ciascuno in PBS/0.5% BSA/0.1% saponina. Rimuovere il tampone di lavaggio e aggiungere 100-300 ml di PBS alle lamelle. NOTA: Per il doppio controcolorazione del corpo, ripetere i passaggi 4,4-4,5 con l&#39;anticorpo primario e secondario ulteriori desiderato in base alle informazioni descritte nella discussione. </li><li> Utilizzando pinze, delicatamente sollevare il vetrino e macchia al largo della PBS utilizzando un tessuto di laboratorio o un tovagliolo di carta. </li><li> Aggiungere 15 ml di un supporto di montaggio anti-sbiadimento adatto contenente 4 &#39;,6-diamidino-2-fenilindolo (DAPI) per il vetrino e invertire il vetrino, celle in basso, su un vetrino da microscopio in vetro. </li><li> Porre i vetrini in una cartella di diapositive e incubare al riparo dalla luce diretta durante la notte a temperatura ambiente per permettere al mezzo anti-sbiadimento difficile impostare curare, secondo le istruzioni del produttore. Una volta che il mezzo anti-dissolvenza è impostato, i vetrini possono essere conservati a 4 ° C o -20 ° C o fino visualizzazione microscopica. </li></ol></li></ol> 5. Visualizzazione microscopica di Coprivetrini Cellule colorate possono essere esposte sia tramite microscopia confocale o un IF in grado microscop verticalee. Sebbene microscopia confocale offre immagini ad alta risoluzione a profondità discrete, un microscopico verticale è in grado di produrre immagini utili più veloce. Per brevità, un metodo di imaging macchiato BE cellule con un microscopio normale è presentato. In generale, all&#39;immagine isotiocianato di fluoresceina (FITC) o fluorofori simili (cioè proteina fluorescente verde), Texas Red e DAPI, richiede un microscopio con filtri in grado di discriminare questi fluorofori. Controllare il microscopio prima di iniziare il protocollo per determinare fluorofori compatibili. <ol><li> Imaging su un IF microscopio standard <ol><li> Rimuovere memorizzati diapositive / vetrini da -20 ° C o 4 ° C e permettere loro di riscaldare a temperatura ambiente. Accendere il microscopio e lampada a vapori di mercurio. </li><li> Posizionare vetrino da microscopio con coprioggetto rivolto verso l&#39;obiettivo sul palco microscopio. Per l&#39;utilizzo di un obiettivo immersione in olio, aggiungere una goccia di olio di immersione sul vetrino. </li><li> Selezionare il filtro DAPI e aprire l&#39;otturatore. </li&gt; <li> Mettere a fuoco l&#39;obiettivo, mentre guardando attraverso gli oculari fino a quando appare l&#39;immagine. Poiché tutte le cellule si colorano con DAPI, i nuclei devono essere facilmente osservabile. </li><li> Raccogliere le immagini utilizzando rispettivo software di elaborazione delle immagini. </li></ol></li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

Abbiamo delineato un metodo per l&#39;applicazione di IF di cellule essere verso chiarire gli effetti fisiologici di terapie mirate su queste cellule. Mentre abbiamo descritto l&#39;uso di queste procedure verso cellule BE, abbiamo trovato che questi metodi sono applicabili ad una varietà di tipi cellulari 13,17,18 anche. Inoltre, queste procedure possono essere modificati in vari modi per ottimizzare la colorazione per target specifici. Troviamo un parametro che ha un effetto di...

Esofago di Barrett (BE) è un cambiamento metaplastica nel normale epitelio squamoso dell&#39;esofago e a seguito di esposizione cronica al contenuto gastrico derivanti da malattia da reflusso gastroesofageo (GERD) 1. BE è pensato per essere un meccanismo protettivo in risposta a GERD, tuttavia, la presenza di BE conferisce un aumentato rischio di adenocarcinoma esofageo (EAC), una malattia che comporta una significativa scarsa sopravvivenza 1. Le stime attuali indicano che fino al 5,6% della popolazione americana hanno essere, tuttavia, come BE è spesso asintomatica, si ritiene che la maggioranza dei BE rimane non diagnosticata 2. Poiché i tassi di incidenza sia di GERD e EAC hanno visto una crescita continua 3, è diventato importante per comprendere i meccanismi molecolari coinvolti nella progressione di BE a EAC, tanto più che queste informazioni potrebbero potenzialmente fornire approcci terapeutici verso la prevenzione della progressione di BE EAC. 1. L&#39;infiammazione cronica, lesioni e danno genotossico derivante dall&#39;esposizione prolungata a contenuto gastrico esercitare una pressione selettiva sulla lesione BE promozione progressione neoplastica di EAC. Un certo numero di alterazioni genetiche sono state identificate durante BE alla progressione EAC. Tuttavia, nonostante questo c&#39;è una netta mancanza di comprensione circa le esatte variazioni di segnalazione cellulare e gli effetti conseguenti su struttura e funzione delle cellule. Immortalata in linee cellulari in vitro sono strumenti utili per studiare gli effetti di segnalazione cellulare, in particolare nei studio degli effetti di composti terapeutici mirati. La recente disponibilità commerciale di linee cellulari immortalizzate BE consente per tali studi. Anche se le analisi high-throughput, come i test di vitalità ampiamente utilizzati, possono essere utili nel valutare gli effetti delle terapie mirate su proliferazione cellulare e survival 4-6, questi test non sono utili per interrogare gli effetti di segnalazione cellulare su morfologia cellulare. Immunofluorescenza (IF) è una tecnica utile per studiare gli effetti delle terapie mirate sulle caratteristiche morfologiche, la crescita e la sopravvivenza delle cellule e le proteine ​​che sono coinvolte. Il nostro laboratorio ha adattato questi metodi verso linee cellulari immortalizzate BE, utilizzando IF per valutare gli effetti di un farmaco clinicamente disponibile su linee cellulari BE, nella speranza di delineare un possibile trattamento chemopreventative e biomarker per il trattamento farmacologico 7. Allo stesso modo, l&#39;applicazione di queste tecniche in EAC, BE e linee cellulari esofagee immortalizzate ha delineato i risultati critici in merito BE di EAC progressione 8-10. Troviamo se l&#39;analisi di cellule trattate con terapie mirate ha valore, consentendo per la caratterizzazione dei cambiamenti nella struttura cellulare e la localizzazione delle proteine. Qui vi presentiamo i nostri metodi di IF di essere cellule immortalizzate per characterize trattamento farmacologico.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of the Material/Equipment</td>
			<td style="width:116px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:224px;">Comments/ Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-A (Metaplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-B (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-C (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-D (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum, Qualified, HI</td>
			<td>Life Technologies</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PSN ANTIBIOTIC MIXTURE</td>
			<td>Life Technologies</td>
			<td>15640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS - Phosphate-Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pro-long Gold Antifade Reagent with DAPI&#160;</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular Glass Coverslip 18mm</td>
			<td>Fisher Scientific</td>
			<td>15-183-86</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-catenin Primary Antibody (Rabbit)</td>
			<td>Cell Signaling</td>
			<td>9562S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexafluor 488 (Goat Anti-Rabbit)</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm TC treated PS dish, sterile</td>
			<td>USA Scientific</td>
			<td>CC7682-3394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-well TC treated PS plate, sterile</td>
			<td>USA Scientific</td>
			<td>5666-5180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin - Fraction V</td>
			<td>Sigma-Aldrich</td>
			<td>85040C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SKI-606 (Bosutinib)</td>
			<td>Selleck Chemicals</td>
			<td>S1014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Square Bioassay Dish&#160;</td>
			<td>Thermo Scientific</td>
			<td>240835</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parafilm&#160;</td>
			<td>VWR</td>
			<td>82024-546</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Pasteur Pipets, Flint Glass</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nexcelom Mini Cell Counter</td>
			<td>Nexcelom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellometer Counting Chambers</td>
			<td>Nexcelom</td>
			<td>CHT4-SD100-014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss Apotome microscope&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an immunofluorescent method for characterization of barrett&#8217;s esophagus cells

Adenocarcinoma esofageo (EAC) ha un tasso di sopravvivenza inferiore al 17% e l&#39;incidenza di EAC è aumentato drammaticamente negli ultimi due decenni. Uno dei fattori di rischio primari di EAC è esofago di Barrett (BE), un cambiamento metaplastica del normale dell&#39;esofago squamoso in risposta al bruciore di stomaco cronica. Nonostante il collegamento consolidata tra EAC e BE, interrogatori degli eventi molecolari, in particolare le vie di segnalazione alterate che coinvolgono la progressione della BE a EAC, sono poco conosciute. Molto di questo è dovuto alla mancanza di idonei modelli in vitro disponibili per studiare queste malattie. Recentemente, BE linee cellulari immortalizzate sono diventati disponibili in commercio che consente per gli studi in vitro di BE. Qui, presentiamo un metodo per immunofluorescenza di linee cellulari immortalizzate BE, permettendo alla caratterizzazione in vitro di segnalamento e struttura cellulare dopo esposizione a composti terapeutici. L&#39;applicazione di queste tecniche sarà help sviluppare comprensione dei meccanismi coinvolti in BE per EAC progressione e fornire potenziali vie per il trattamento e la prevenzione di EAC.

Un esempio dei risultati ottenuti dall&#39;applicazione delle procedure descritte è illustrato nelle figure 1A-D. Coprioggetto con CP-D e BE cellule CP-C sono stati trattati per 24 ore con 1 mM dell&#39;inibitore Src famiglia, SKI-606, o di un veicolo (DMSO) e colorati con le modalità di cui sopra per il adherens Junction e Wnt segnalazione proteine, β -catenina. Visualizzazione di β-catenina è stata ottenuta mediante l&#39;etichettatura con IgG anti-coniglio accoppiato ad un fluorocromo verde, men...

Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of Barrettâ€™s Esophagus Cells at JoVE.com

An Immunofluorescent Method for Characterization of Barrett&amp;#8217;s Esophagus Cells

Vi è la necessità di migliorare discernibile informazioni sui driver molecolari dell&#39;esofago di Barrett. Immunofluorescenza è una tecnica utile per comprendere gli effetti di segnalazione cellulare sulla morfologia cellulare. Presentiamo un protocollo semplice ed efficace per l&#39;uso di immunofluorescenza per valutare il trattamento terapeutico nelle cellule esofago di Barrett.

Un metodo di immunofluorescenza per la caratterizzazione di cellule esofago di Barrett

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

an-immunofluorescent-method-for-characterization-barretts-esophagus

Research

JoVE Journal

Biology

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

14.0K Views.  St. Joseph's Hospital and Medical Center.  Vi è la necessità di migliorare discernibile informazioni sui driver molecolari dell'esofago di Barrett. Immunofluorescenza è una tecnica utile per comprendere gli effetti di segnalazione cellulare sulla morfologia cellulare. Presentiamo un protocollo semplice ed efficace per l'uso di immunofluorescenza per valutare il trattamento terapeutico nelle cellule esofago di Barrett. 

Video: An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

Un metodo per la produzione di proteine ​​ricombinanti mCD1d in cellule di insetto.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Ricerca

Biologia

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Un metodo per la coltura embrionale<em&gt; C. elegans</em&gt; Celle

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Un metodo innovativo per Exosome quantificazione e Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Isolamento di miofibroblasti da mouse e Esofago umano

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Miglioramento Tecnica Swiss-laminazione per intestinale Preparazione del tessuto per immunoistochimica e analisi immunofluorescenza

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Un metodo facile per piante polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

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

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

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

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

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

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

In Situ Immunofluorescenti colorazione di autofagia nelle cellule staminali muscolari

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.