Este trabalho foi financiado por doações do St, Fundação de Joseph (AJF, LJI) e American Lung Association, RG-224607-N (LJI).

1. Linha celular Manutenção <ol><li> Humana imortalizada BE displásicas de alto grau (CP-B, CP-C, CP-D) e metaplásico (CP-A) de linhas celulares por transfecção estável da telomerase humana transcriptase (TERT) de proteína 11 reversa. </li><li> Manter BE linhas de células de queratinócitos em meio suplementado com extracto de pituitária de bovino (BPE), factor de crescimento epidérmico (EGF), 5% de soro fetal bovino (FBS), aminoácidos não essenciais (NEAA), penicilina-estreptomicina-neomicina (PSN), e padrão condições de cultura de tecidos (37 ° C, 5% de CO 2, 98% de humidade). </li></ol> 2. BE celular chapeamento <ol><li> Preparação de 12 poços de placas / lamelas. Coloque 18 milímetros lamínulas em uma placa de Petri e autoclave de vidro. Transferência autoclavada 18 milímetros lamelas de vidro em um prato de 12 poços de cultura de tecido utilizando uma pipeta de vidro estéril ligada a um tubo de vácuo. Lave as lamelas com PBS estéril e mídia. </li><li> Trypsinize e contagem de células ser imortalizado. Contar células using contador de células automatizado ou um hemocitômetro. Diluir as células em meio de cultura a uma concentração de 20.000-40.000 células / ml. </li><li> Colocar 1 ml de células em cada cavidade contendo uma lamela para uma contagem de células totais de 20.000-40.000 células / poço. </li><li> Utilizar uma ponta de pipeta estéril de empurrar suavemente a lamela para o fundo do poço, para assegurar que as células fixar a lamela. </li></ol> 3. Tratamento de Drogas de Células BE <ol><li> Diluir fármaco seleccionado em meio morno (37 ° C) até à concentração desejada e misturar bem, invertendo o tubo várias vezes ou usando um misturador de vórtice. As concentrações de medicamento determinado empiricamente. Comece o tratamento 24 horas após a semeadura inicial das células ser o de permitir que as células de fixação para a lamela </li><li> Para efeito de comparação e controle, o tratamento de uma lamela adicional de células BE com 0,1% de veículo (dimetilsulfóxido [DMSO] ou agente usado para solubilizar composto) diluído em meios de cultura. Placa de etiqueta com uma caneta de laboratório para identificar a droga e veculo amostras tratadas. Colocar as células em uma incubadora de cultura de células definido como 37 ° C, 5% de CO 2, 98% de humidade e incubar durante pontos de tempo desejados. </li></ol> 4. Immunofluorescent Coloração <ol><li> Fixação <ol><li> Seguindo o tempo de incubação desejado do tratamento com droga, os meios de aspiração e lava-se rapidamente as lamelas com tampão fosfato salino 1x (PBS) à temperatura ambiente (TA, 25 ° C). </li><li> Aspirar o PBS e adicionar 1 mL de PBS contendo 4% de PFA [4% PFA / PBS] a cada poço e incube à temperatura ambiente durante 30 min. </li><li> Aspirar a 4% PFA / PBS e lavar o lamelas 3x, 5 min cada com PBS à temperatura ambiente. Armazene lamelas para uma semana em PBS/0.1% PFA a 4 ° C, se desejar. </li></ol></li><li> Permeabilização / Bloqueio Permeabilizar células PFA fixas para permitir que o anticorpo para aceder aos alvos intracelulares. Para extracelular surgiu proteínas, executar todos os passos subsequentes sem saponina adicionado à solução de BSA 1x PBS/0.5%. <ol><li> Reduzir o fundo por incubação com 50 mM de NH 4 Cl diluído em PBS durante 10 min à temperatura ambiente e lava-se com PBS, uma vez RT durante 5 min. </li><li> Incubar ser células durante 30 minutos em 100-300 ul de PBS contendo 0,1% de saponina e 0,5% de albumina de soro bovino (BSA). Dilui-se a partir de 10% de saponina estoque, esterilizada por filtração, preparada em água e armazenado a 4 ° C. </li></ol> Nota: Isto impede a ligação não específica de anticorpos contra proteínas celulares, bloqueando lamelas, devido à adição de 0,5% de BSA. Alternativamente, substituem BSA com soro de cabra diluído a 5% para ajudar a reduzir a ligação não específica de anticorpos. </li><li> Preparação de secção Humano <ol><li> Prepare um prato quadrado bioensaio por camadas na parte inferior da câmara com papel-toalha molhada e coloque uma camada de Parafilm no topo. </li><li> Gentilmente transferir as lamelas, células voltadas para cima, para o Parafilm usando uma pinça e uma agulha de seringa. </li><li> Marque o Parafilm com uma caneta de laboratório para identificar lamelas. </li></ol></li><li>Primário Incubação de anticorpos <ol><li> Dilui-se o anticorpo primário em PBS contendo BSA a 0,5% tanto e 0,1% de saponina. </li><li> Seque levemente para fora da solução de bloqueio utilizando um tecido de laboratório e adicionar 100 ul de solução de anticorpo primário. Incubar o anticorpo primário às células BE durante a noite a 4 ° C. </li></ol></li><li> A incubação do anticorpo secundário <ol><li> Lavar as lâminas 3x com PBS/0.5% BSA/0.1% de saponina por 5 min cada em temperatura ambiente. </li><li> Dilui-se o anticorpo secundário conjugado com um marcador fluorescente em PBS/0.5% BSA/0.1% de saponina que o anticorpo secundário reconhece as espécies em que o anticorpo primário foi levantadas. </li><li> Adicionar 100 ul de anticorpo diluído em cada lamela. Incubar o anticorpo secundário durante 2 horas à temperatura ambiente. </li></ol></li><li> Lavagem e montagem de Lamelas <ol><li> Lave as lamelas de 3x, 5 min cada em PBS/0.5% BSA/0.1% de saponina. Remover o tampão de lavagem e adicionar 100-300 ul de PBS para as lamelas. NOTA: Para a dupla anticoloração do corpo, repita os passos de 4,4-4,5 com o anticorpo primário e secundário adicional desejado de acordo com informações descritas na discussão. </li><li> Utilizando uma pinça, retire cuidadosamente a lamela e secar fora da PBS usando um tecido de laboratório ou toalha de papel. </li><li> Adicionar 15 mL de um meio de montagem anti-fade apropriado contendo 4 &#39;,6-diamidino-2-phenylindole (DAPI) para a lamela e inverter a lamela, células para baixo, sobre uma lâmina de vidro. </li><li> Colocar as lâminas em uma pasta de slides e incubar longe da luz direta durante a noite à temperatura ambiente para permitir que o meio anti-fade definir difícil de curar, de acordo com as instruções do fabricante. Uma vez que o meio anti-desbotamento definiu, as lâminas podem ser armazenadas a 4 ° C ou -20 ° C até a visualização microscópica. </li></ol></li></ol> 5. Visualização microscópica de Lamelas Células coradas podem ser visualizados através de qualquer microscopia confocal ou um IF microscop vertical capaze. Apesar de microscopia confocal fornece imagens de alta resolução em profundidades discretas, uma microscópica vertical é capaz de produzir imagens úteis mais rápido. Para resumir, um método de imagem manchado BE células usando microscopia padrão é apresentado. Em geral, a imagem de isotiocianato de fluoresceína (ITCF) ou fluoróforos semelhantes (isto é, proteína fluorescente verde), Texas Red e DAPI, requer um microscópio com filtros capazes de discriminar estes fluoróforos. Confira o microscópio antes de iniciar o protocolo para determinar fluoróforos compatíveis. <ol><li> Imagem em um IF Microscópio Padrão <ol><li> Retirar armazenados slides / lamelas de -20 ° C ou 4 ° C e permitem-lhes a aquecer até à temperatura ambiente. Ligue microscópio e lâmpada de arco de mercúrio. </li><li> Coloque lâmina de microscópio com lamela voltada para objetivo para o palco microscópio. Para o uso de um objetivo de imersão em óleo, adicione uma gota de óleo de imersão sobre lamela. </li><li> Selecione o filtro DAPI e abrir o obturador. </li&gt; <li> Concentre-se o objetivo ao olhar pelas oculares até que a imagem apareça. Uma vez que todas as células que coram com DAPI, os núcleos devem ser facilmente observável. </li><li> Coletar imagens usando o respectivo software de processamento de imagem. </li></ol></li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Temos delineado um método para aplicação de IF de células ser no sentido de elucidar os efeitos fisiológicos de terapias específicas sobre estas células. Embora se tenha descrito o uso desses procedimentos contra células BE, verificou-se que estes métodos também são aplicáveis ​​a uma variedade de diferentes tipos de células 13,17,18. Além disso, esses procedimentos podem ser modificados de várias formas para optimizar a coloração para alvos específicos. Nós...

Esôfago de Barrett (EB) é uma mudança metaplásico no epitélio escamoso normal do esôfago e uma consequência da exposição crônica ao conteúdo gástricas resultantes da doença do refluxo gastroesofágico (DRGE) 1. BE é pensado para ser um mecanismo de proteção em resposta a RGE, no entanto, a presença de BE confere um risco aumentado de adenocarcinoma esofágico (EAC), uma doença que acarreta uma sobrevivência significativamente pobre 1. As estimativas atuais sugerem que até 5,6% da população americana tem BE, porém, como BE é muitas vezes assintomática, pensa-se que a maioria das BE permanece sem diagnóstico 2. Como as taxas de incidência de ambos DRGE e EAC têm visto um crescimento contínuo 3, tornou-se importante para compreender os mecanismos moleculares envolvidos na progressão do BE para EAC, particularmente porque essas informações poderiam fornecer abordagens terapêuticas no sentido de prevenir a progressão da BE à EAC. 1. A inflamação crônica, lesões e danos genotóxicos resultantes da exposição prolongada ao conteúdo gástrico exercer pressão seletiva sobre a lesão estar promovendo a progressão neoplásica a EAC. Uma série de alterações genéticas foram identificadas durante a BE à progressão EAC. No entanto, apesar disso, há uma clara falta de entendimento sobre as mudanças exatas na sinalização celular e os efeitos subsequentes sobre a estrutura e função celular. Imortalizado em linhas celulares in vitro são ferramentas úteis para o estudo dos efeitos de sinalização de células, particularmente em investigar os efeitos dos compostos terapêuticos alvo. A recente disponibilidade comercial de ser linhas de células imortalizadas permite tais estudos. Embora os ensaios de alto rendimento, como os ensaios de viabilidade amplamente utilizada, pode ser valioso para avaliar os efeitos das terapias-alvo sobre a proliferação celular e survival 4-6, estes ensaios não são úteis para interrogar os efeitos de sinalização de células sobre a morfologia celular. Coloração de imunofluorescência (IF) é uma técnica útil para investigar os efeitos de terapias específicas sobre as características morfológicas, de crescimento e sobrevivência das células e as proteínas que estão envolvidas. Nosso laboratório adaptou esses métodos para linhas de células imortalizadas BE, usando IF para avaliar os efeitos de uma droga clinicamente disponíveis mediante linhas de células BE na esperança de delinear um possível tratamento quimiopreventivo e biomarcador para tratamento de drogas 7. Da mesma forma, a aplicação destas técnicas na EAC, BE e linhas de células do esôfago imortalizados delineou conclusões críticas sobre BE à progressão EAC 8-10. Encontramos se a análise de células ser tratados com terapias direcionadas tem valor, permitindo a caracterização de mudanças na estrutura celular e localização de proteínas. Aqui, apresentamos os nossos métodos para IF de ser células imortalizadas para characterize tratamento medicamentoso.

<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 de esôfago (EAC) tem uma taxa de sobrevida global de menos de 17% ea incidência de EAC aumentou dramaticamente ao longo das últimas duas décadas. Um dos principais fatores de risco de EAC é o esôfago de Barrett (BE), uma mudança metaplásico do esôfago escamoso normal em resposta a azia crônica. Apesar da ligação bem estabelecida entre EAC e BE, o interrogatório dos eventos moleculares, particularmente vias de sinalização alterados envolvendo progressão da SER para EAC, são mal compreendidos. Grande parte isto é devido à falta de modelos in vitro adequados disponíveis para estudar estas doenças. Recentemente, as linhas de células imortalizadas BE tornaram-se disponíveis comercialmente permitindo estudos in vitro de BE. Aqui, apresentamos um método para coloração de imunofluorescência de linhas de células imortalizadas BE, permitindo caracterização in vitro de sinalização e estrutura da célula após a exposição a compostos terapêuticos. Aplicação destas técnicas irá help desenvolver insights sobre os mecanismos envolvidos na BE à progressão EAC e fornecer potenciais caminhos para o tratamento e prevenção da EAC.

Um exemplo dos resultados obtidos com a aplicação dos procedimentos descritos está ilustrada nas Figuras 1A-D. Lamelas com PC-D e FP-C BE células foram tratadas durante 24 horas com 1 uM do inibidor de Src família, SKI-606, ou veículo (DMSO) e coradas utilizando os procedimentos descritos acima para o Adherens junção de sinalização Wnt e proteína, β -catenina. Visualização de β-catenina foi conseguida por marcação com uma IgG anti-coelho acoplado a um fluoróforo verde, enquanto que a r...

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

Há uma necessidade visível de uma melhor informação sobre os condutores moleculares de Esôfago de Barrett. Coloração de imunofluorescência é uma técnica útil para entender os efeitos da sinalização celular na morfologia celular. Nós apresentamos um protocolo simples e eficaz para o uso de coloração de imunofluorescência para avaliar tratamento terapêutico em células esôfago de Barrett.

O método de imunofluorescência para Caracterização de Células Esôfago 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 ...

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

Research

JoVE Journal

Biology

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

14.0K visualizações.  St. Joseph's Hospital and Medical Center.  Há uma necessidade visível de uma melhor informação sobre os condutores moleculares de Esôfago de Barrett. Coloração de imunofluorescência é uma técnica útil para entender os efeitos da sinalização celular na morfologia celular. Nós apresentamos um protocolo simples e eficaz para o uso de coloração de imunofluorescência para avaliar tratamento terapêutico em células esôfago de Barrett. 

Vídeo: 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.

Um método para produção de proteína recombinante em células de inseto mCD1d.

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

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.

Um Método para o cultivo embrionário<em&gt; C. elegans</em&gt; Células

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.

Um método inovador para exossomo Quantificação 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 de Miofibroblastos de Mouse e esôfago humano

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.

Técnica Swiss-rolamento melhorado para preparo intestinal tecido por imuno-histoquímica e análises de imunofluorescência

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.

Um método fácil para a planta 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.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

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.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

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.

Coloração imunofluorescente in situ de autofagia em células estaminais musculares

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.