Ce travail a été soutenu par des subventions du St, la Fondation Joseph (AJF, LJI) et l&#39;American Lung Association, RG-224607-N (LJI).

1. Entretien Cell ligne <ol><li> Humaine immortalisée BE dysplasiques de haut grade (CP-B, CP-C, CP-D) et métaplasique (CP-A) des lignées de cellules par transfection stable de la télomérase humaine transcriptase inverse (TERT) protéine 11. </li><li> Maintenir BE lignées cellulaires dans un milieu de kératinocytes supplémenté avec de l&#39;extrait de bovin pituitaire (BPE), le facteur de croissance épidermique (EGF), 5% de sérum bovin fœtal (FBS), acides aminés non essentiels (NEAA), de la pénicilline-streptomycine-néomycine (PSN), et la norme conditions de culture de tissus (37 ° C, 5% CO 2, 98% d&#39;humidité). </li></ol> 2. ÊTRE cellulaire Plaquage <ol><li> Préparation de 12 puits plaque / lamelles. Placez les lamelles 18 mm dans un plat et autoclave Petri verre. Transfert autoclavé 18 lamelles de verre mm dans une boîte de culture tissulaire à 12 puits en utilisant une pipette en verre stérile fixée à un tube à vide. Laver la lamelle avec du PBS stérile et médias. </li><li> Trypsiniser et compter les cellules d&#39;être immortalisé. Compter les cellules using un compteur de cellules automatisé ou un hémocytomètre. Diluer les cellules dans un milieu de culture à une concentration de 20.000-40.000 cellules / ml. </li><li> Placer 1 ml de cellules dans chaque puits contenant une lamelle couvre-objet pour un nombre total de cellules de 20.000-40.000 cellules / puits. </li><li> Utilisation d&#39;une pointe de pipette stérile pour pousser doucement la lamelle au fond du puits afin d&#39;assurer que les cellules se fixent à la lamelle couvre-objet. </li></ol> 3. Traitement de la toxicomanie de cellules soient <ol><li> Diluer médicament sélectionné dans (37 ° C) médias chauds à la concentration souhaitée et bien mélanger en inversant le tube à plusieurs reprises ou en utilisant un mélangeur vortex. Concentrations de médicament déterminée empiriquement. Commencer le traitement 24 heures après l&#39;ensemencement initial des cellules BE pour permettre aux cellules de fixation sur la lamelle </li><li> A titre de comparaison et de contrôle, traiter une lamelle supplémentaire de cellules de BE avec 0,1% de véhicule (diméthylsulfoxyde [DMSO] ou agent utilisé pour solubiliser composé) dilué dans un milieu de culture. plaque d&#39;étiquettes avec un stylo de laboratoire pour identifier le médicament et vecule échantillons traités. Placez les cellules dans un incubateur de culture cellulaire mis à 37 ° C, 5% de CO 2, 98% d&#39;humidité et incuber pendant des moments souhaités. </li></ol> 4. Immunofluorescence <ol><li> Fixation <ol><li> A la suite du temps d&#39;incubation désirée du traitement médicamenteux, aspirer le support et laver les lamelles rapidement avec 1x solution saline tamponnée phosphate (PBS) à température ambiante (TA, 25 ° C). </li><li> Aspirer le PBS et ajouter 1 ml de PBS contenant 4% de PFA [PFA à 4% / PBS] à chaque puits et incuber à température ambiante pendant 30 min. </li><li> Aspirer le PFA 4% / PBS et laver le couvre-3x, 5 min chacun avec PBS à température ambiante. lamelles de magasins pour une semaine en PBS contenant 0,1% de PFA à 4 ° C si vous le souhaitez. </li></ol></li><li> Perméabilisation / blocage Perméabiliser les cellules PFA fixes pour permettre à l&#39;anticorps pour accéder aux cibles intracellulaires. Pour extracellulaire surface des protéines, effectuer toutes les étapes ultérieures sans saponine ajouté à la solution de BSA 1x PBS/0.5 de%. <ol><li> Réduire le fond par incubation avec 50 mM de NH 4 Cl dilué dans du PBS pendant 10 min à température ambiante et laver avec du PBS une fois que la température ambiante pendant 5 min. </li><li> Incuber être des cellules pendant 30 min à 100 à 300 pi de PBS contenant 0,1% de saponine et 0,5% d&#39;albumine de sérum bovin (BSA). Diluer la saponine de filtre stérilisé 10% stock, préparé dans l&#39;eau et conservé à 4 ° C. </li></ol> NOTE: Ceci empêche la liaison non spécifique des anticorps dirigés contre des protéines cellulaires par des lamelles de blocage dus à l&#39;addition de 0,5% de BSA. En variante, substituer BSA avec du sérum de chèvre dilué à 5% pour réduire la liaison non spécifique d&#39;anticorps. </li><li> Préparation de la Chambre humain <ol><li> Préparer un plat carré d&#39;essai biologique en superposant le fond de la chambre avec des serviettes en papier humide et placez une couche de Parafilm sur le dessus. </li><li> Transférer délicatement les lamelles, les cellules orientées vers le haut, sur le Parafilm aide d&#39;une pince et une aiguille de seringue. </li><li> Marquer le Parafilm avec un stylo de laboratoire pour identifier les lamelles. </li></ol></li><li>Anticorps primaire incubation <ol><li> Diluer l&#39;anticorps primaire dans du PBS contenant à la fois 0,5% de BSA et 0,1% de saponine. </li><li> Épongez délicatement la solution de blocage à l&#39;aide d&#39;un tissu de laboratoire et ajouter 100 ul de la solution d&#39;anticorps primaire. Incuber l&#39;anticorps primaire sur les cellules de BE pendant une nuit à 4 ° C. </li></ol></li><li> Incubation de l&#39;anticorps secondaire <ol><li> Laver les lames 3x avec PBS/0.5% BSA/0.1% de saponine pour 5 min chacune à la température ambiante. </li><li> Diluer l&#39;anticorps secondaire conjugué à un marqueur fluorescent dans PBS/0.5% BSA/0.1% de saponine que l&#39;anticorps secondaire reconnaît l&#39;espèce dans laquelle l&#39;anticorps primaire a été soulevée. </li><li> Ajouter 100 ul d&#39;anticorps dilué à chaque lamelle. Incuber l&#39;anticorps secondaire pendant 2 h à température ambiante. </li></ol></li><li> Laver et montage de lamelles couvre- <ol><li> Laver le couvre-3x, 5 min chacun dans PBS/0.5% BSA/0.1% de saponine. Éliminer le tampon de lavage et ajouter 100 à 300 pi de PBS aux lamelles couvre-objet. REMARQUE: Pour une double lutte contrecoloration du corps, répétez les étapes 4.4 à 4.5 avec l&#39;anticorps primaire et secondaire supplémentaire souhaitée selon les modalités détaillées dans la discussion. </li><li> En utilisant des pinces, soulever délicatement la lamelle et éponger la PBS en utilisant un tissu de laboratoire ou une serviette en papier. </li><li> Ajouter 15 ul d&#39;un milieu de montage anti-décoloration approprié contenant 4 &#39;,6-diamidino-2-phénylindole (DAPI) pour inverser la lamelle couvre-objet et la lamelle couvre-objet, des cellules vers le bas, sur une lame de microscope en verre. </li><li> Placer les lames dans un dossier de la lame et incuber abri de la lumière directe du jour au lendemain à la température ambiante pour permettre le support anti-décoloration dur mis à guérir, selon les instructions du fabricant. Une fois le support anti-décoloration a mis, diapositives peuvent être conservées à 4 ° C ou -20 ° C jusqu&#39;à ce que la visualisation microscopique. </li></ol></li></ol> 5. Visualisation microscopique des lamelles couvre- Les cellules colorées peuvent être visualisés par l&#39;intermédiaire soit microscopie confocale ou un SI microscop verticale capablee. Bien que la microscopie confocale fournit des images haute résolution à des profondeurs discrètes, un microscopique verticale est capable de produire des images utiles plus rapidement. Par souci de concision, un procédé d&#39;imagerie souillé BE cellules par microscopie norme est présentée. En général, à l&#39;image de l&#39;isothiocyanate de fluorescéine (FITC) ou similaires fluorophores (par exemple la protéine verte fluorescente), le rouge Texas et DAPI, nécessite un microscope avec des filtres capables de discriminer ces fluorophores. Vérifiez le microscope avant de commencer le protocole pour déterminer fluorophores compatibles. <ol><li> Imagerie sur un microscope standard IF <ol><li> Retirer stockées diapositives / lamelles couvre-objet à partir de -20 ° C ou à 4 ° C et leur permettre de se réchauffer jusqu&#39;à la température ambiante. Mise sous tension et microscope lampe à arc au mercure. </li><li> Placez lame de microscope avec lamelle orientée vers l&#39;objectif sur la platine du microscope. Pour l&#39;utilisation d&#39;un objectif à immersion d&#39;huile, ajouter une goutte d&#39;huile d&#39;immersion sur la lamelle. </li><li> Sélectionnez le filtre DAPI et ouvrir l&#39;obturateur. </li&gt; <li> Concentrer l&#39;objectif tout en regardant à travers les oculaires jusqu&#39;à ce que l&#39;image apparaît. Comme toutes les cellules se colorent au DAPI, les noyaux doivent être facilement observable. </li><li> Recueillir des images en utilisant un logiciel de traitement d&#39;image respective. </li></ol></li></ol>

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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Nous avons décrit une méthode pour l&#39;application de l&#39;IF de cellules de BE à élucider les effets physiologiques des thérapies ciblées sur ces cellules. Alors que nous avons décrit l&#39;utilisation de ces procédures à l&#39;égard des cellules de BE, nous avons constaté que ces procédés sont également applicables à une variété de types cellulaires différents 13,17,18. En outre, ces procédures peuvent être modifiées de plusieurs façons d&#39;optimiser coloration pour des objectifs ...

L&#39;oesophage de Barrett (BE) est un changement de métaplasie de l&#39;épithélium malpighien normal de l&#39;œsophage et une conséquence de l&#39;exposition chronique à du contenu gastrique résultant de la maladie de reflux gastro-œsophagien (RGO) 1. BE est pensé pour être un mécanisme de protection en réponse à la DIRD, mais la présence de BE confère un risque accru d&#39;adénocarcinome de l&#39;œsophage (EAC), une maladie qui porte un faible taux de survie significativement 1. Les estimations actuelles indiquent que jusqu&#39;à 5,6% de la population américaine ont BE, mais comme BE est souvent asymptomatique, on pense que la majorité des BE reste non diagnostiquée 2. Comme les taux des deux RGO et EAC incidence ont connu une croissance continue 3, il est devenu important de comprendre les mécanismes moléculaires impliqués dans la progression de BE à EAC, surtout en ce qui pourrait fournir des approches thérapeutiques vers la prévention de la progression de la BE à l&#39;EAC. 1. L&#39;inflammation chronique, des blessures et des dommages génotoxiques résultant d&#39;une exposition prolongée à contenu gastrique exercent une pression sélective sur la lésion BE promouvoir la progression néoplasique à CAE. Un certain nombre de modifications génétiques ont été identifiées pendant BE à la progression de la CAE. Cependant, malgré cela, il ya un manque de compréhension des changements exacts dans la signalisation cellulaire et les effets subséquents sur la structure et la fonction des cellules. Immortalisées dans des lignées cellulaires in vitro sont des outils utiles pour étudier les effets de la signalisation cellulaire, en particulier dans l&#39;étude des effets des composés thérapeutiques ciblés. La récente disponibilité commerciale de BE lignées cellulaires immortalisées permet à de telles études. Bien tests à haut débit, tels que les tests de viabilité largement utilisés, peuvent être utiles pour évaluer les effets des thérapies ciblées sur la prolifération cellulaire et survival 4-6, ces dosages ne sont pas utiles pour interroger les effets de la signalisation cellulaire sur la morphologie des cellules. Immunofluorescence (IF) est une technique utile pour étudier les effets des thérapies ciblées sur les caractéristiques morphologiques, la croissance et la survie des cellules et des protéines qui sont impliquées. Notre laboratoire a adapté ces méthodes vers des lignes BE cellulaires immortalisées, en utilisant SI pour évaluer les effets d&#39;un médicament cliniquement disponibles sur des lignées cellulaires de BE dans l&#39;espoir de délimiter un éventuel traitement chimiopréventifs et biomarqueur pour le traitement de la drogue 7. De même, l&#39;application de ces techniques dans la CAE, BE et les lignées cellulaires immortalisées de l&#39;œsophage a délimité conclusions critiques concernant BE à la progression de la CAE 8-10. Nous trouver si l&#39;analyse de cellules être traités avec des thérapies ciblées a de la valeur, ce qui permet pour la caractérisation des changements dans la structure de la cellule et la localisation des protéines. Ici, nous présentons nos méthodes de SI de immortalisées être des cellules à characterize traitement de la toxicomanie.

<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

Adénocarcinome oesophagien (EAC) a un taux de survie global de moins de 17% et l&#39;incidence de la CAE a augmenté de façon spectaculaire au cours des deux dernières décennies. L&#39;un des principaux facteurs de risque de la CAE est l&#39;oesophage de Barrett (BE), un changement de métaplasie épidermoïde de l&#39;œsophage normale en réponse à des brûlures d&#39;estomac chroniques. Malgré la connexion bien établie entre CAE et BE, interrogation des événements moléculaires, en particulier les voies de signalisation altérées impliquant progression de BE à EAC, sont mal compris. Beaucoup de ceci est dû à l&#39;absence d&#39;convenable dans des modèles in vitro disponibles pour étudier ces maladies. Récemment, les lignes BE cellulaires immortalisées sont devenus disponibles dans le commerce permettant des études in vitro de BE. Ici, nous présentons une méthode de coloration par immunofluorescence de lignes BE cellulaires immortalisées, permettant la caractérisation in vitro de la signalisation cellulaire et de la structure après exposition à des composés thérapeutiques. L&#39;application de ces techniques se help développer comprendre les mécanismes impliqués dans la progression BE EAC et de fournir des pistes potentielles pour le traitement et la prévention de l&#39;EAC.

Un exemple des résultats obtenus à partir de l&#39;application des procédures décrites, est illustrée sur les figures 1A-D. Lamelles couvre avec CP-D et cellules BE CP-C ont été traités pendant 24 heures avec 1 uM de l&#39;inhibiteur de Src de la famille, SKI-606, ou d&#39;un véhicule (DMSO) et colorées en utilisant les procédures ci-dessus pour la jonctions adhérentes et signalisation Wnt protéine, β -caténine. Visualisation de β-caténine a été obtenue par marquage avec un anticorps ...

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

Il est nécessaire d&#39;améliorer l&#39;information visible sur les moteurs moléculaires de l&#39;oesophage de Barrett. Immunofluorescence est une technique utile pour comprendre les effets de la signalisation cellulaire sur la morphologie cellulaire. Nous présentons un protocole simple et efficace pour l&#39;utilisation de la coloration par immunofluorescence pour évaluer le traitement thérapeutique dans les cellules oesophage de Barrett.

Une méthode d&#39;immunofluorescence pour la caractérisation des cellules oesophage de 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 ...

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Research

JoVE Journal

Biology

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

14.0K vues.  St. Joseph's Hospital and Medical Center.  Il est nécessaire d'améliorer l'information visible sur les moteurs moléculaires de l'oesophage de Barrett. Immunofluorescence est une technique utile pour comprendre les effets de la signalisation cellulaire sur la morphologie cellulaire. Nous présentons un protocole simple et efficace pour l'utilisation de la coloration par immunofluorescence pour évaluer le traitement thérapeutique dans les cellules oesophage de Barrett. 

Vidéo: 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 procédé de production de protéine recombinante mCD1d en cellules d&#39;insectes.

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

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Biologie

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 procédé de culture embryonnaire<em&gt; C. elegans</em&gt; Cellules

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.

Une méthode innovante pour Exosome Quantification et Taille Mesure

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.

Isolement de myofibroblastes de Mouse et œsophage humain

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.

Amélioration Technique Swiss-laminage pour la préparation Intestinal des tissus pour immunohistochimique et immunofluorescence Analyses

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.

Une méthode facile pour des plantes polysomes 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.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

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.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

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.

Coloration immunofluorescente in situ de l&#39;autophagie dans les cellules souches musculaires

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.