Este trabalho foi apoiado por um dos Institutos Nacionais de Saúde Grant DK083327 e DK093491 (a SZH).

1. Preparando células pancreáticas acinares para Imagem de cálcio <ol><li> Preparar tampão de incubação contendo HEPES a 20 mM HEPES, 95 mM de NaCl, 4,7 mM KCl, 0,6 mM MgCl 2, 1,3 mM de CaCl2, 10 mM de glucose, 2 mM de glutamina e 1 × aminoácidos não essenciais do meio mínimo de Eagle. Ajuste da solução final a pH 7,4 com NaOH. </li><li> Preparar um tampão de incubação de BSA por adição de BSA (1% w / v final) a 25 ml de tampão de incubação de HEPES (descrito acima). </li><li> Preparar um tampão de digestão de colagenase por adição de 1,1 mg / mL (200 unidades / ml), colagenase tipo 4 e 1 mg / ml de inibidor de tripsina de soja a 6 ml de tampão de incubação de BSA (descrito acima). </li><li> Ácido-lavagem mm lamelas 22x22 por imersão em duas partes HNO 3 e uma parte de HCl por 2 horas. Decantar com água DI e armazenar em etanol 70%. </li><li> Sacrificá um rato por asfixia CO 2. Orientar o animal em decúbito dorsal, e preparar a superfície abdominal por cledesmame com etanol a 70%. Realizar uma laparotomia para expor a cavidade abdominal. Dissecar o pâncreas e lave imediatamente em um pequeno barco pesar contendo 6 ml de tampão de digestão colagenase. </li><li> Dissecar afastado qualquer grandes pedaços de vasos sanguíneos de gordura ou visível, em seguida, retire 5 ml de tampão de digestão colagenase e adicioná-lo de volta para o tubo cônico de 15 ml. </li><li> Com uma tesoura de dissecção fina picar o pâncreas no pequeno barco pesando contendo 1 ml de tampão de digestão colagenase. Tritura até que a solução resultante aparece uniformemente dispersas. </li><li> Transferir o produto triturado para um frasco de plástico ml de Erlenmeyer de 125, e adicionar os restantes 5 ml de tampão de digestão de colagenase para o recipiente. Certifique-se de que o tecido é completamente submerso em tampão. </li><li> Colocar o balão num banho de água a 37 ° C e agitar a 90 rpm por 30 min. Durante este curto período de tempo de inatividade, preparar agonistas ativação de cálcio (por exemplo ceruleina, carbacol) e lavar o mm capa ácido lavado 22x22desliza com água DI. A seco, e, em seguida, colocar em cima de uma superfície plana, revestida por filme de laboratório. </li><li> Após 30 min a digerir está completa, transferir a suspensão de volta para o tubo de 15 ml cónica e permitir que as células assentar. Pipetar cuidadosamente o tampão de digestão colagenase e substituir com 6 ml de tampão de incubação BSA. Agitar vigorosamente o tubo à mão durante 10 segundos, a fim de dispersar as células em agrupamentos mais pequenos. Imediatamente após a agitação, retirar qualquer grande, detritos flutuantes da mídia não liquidados. </li><li> Permitir que as células restantes para resolver, e trocar os meios de comunicação com tampão de incubação fresco BSA. </li><li> Repetir o passo 1.11, duas vezes, até a suspensão é composta por apenas pequenos aglomerados que só são finamente visível a olho nu. As células devem se assemelham aos descritos na Figura 1A (linha superior). </li><li> Remover o tampão de incubação de BSA e HEPES substituir com tampão de incubação. </li><li> Prepare um 750 mM, estoque 100X de Fluo-04:00 em 10% DMSO. </li><li> Carregar as células no tubo cónico de 15 ml contendo a suspensão de células e o tampão de incubação de BSA livre de HEPES. Para fazer isso, adicionar um volume apropriado (diluição 1:100) da solução à suspensão de células. Então placa 500 ul desta suspensão em cada lamela 22x22 mm. </li><li> Mantenha lamelas no escuro à temperatura ambiente. Dê o primeiro Ca 2 + medições 30 min após o carregamento. O corante é automaticamente removida do meio após o início da perfusão, o que deve ocorrer após 30 min de carga de corante. </li></ol> 2. Medição de cálcio em células pancreáticas acinares <ol><li> Lavar cada perfusão set-up com água DI e preencha com uma quantidade adequada de tampão ou agonista. Primeiro cada seringa com tampão ou agonista para garantir o fluxo adequado. Grampo seringas a um anel de ficar cerca de 1-2 metros acima do palco microscópio. </li><li> Use uma pinça fina para agarrar com cuidado a partir de sua borda uma suspensão de células contendo lamela. Inclinar a tampaescorregar 45 °, e permitir que o excesso de tampão para fugir. Coloque a lamela sobre a vedação de borracha com as células na parte superior da lamela e montar a câmara (Figuras 2A e 2B). </li><li> Fixar a câmara de perfusão para a fase e inserir o tubo que contém tampão para a primeira entrada da câmara (Figura 2C). Rodar a seringa contendo tampão e permitir que o tampão para perfundir sobre toda a superfície da lamela. Uma vez que o tampão tenha atingido a extremidade oposta da câmara, inserir uma linha de vácuo para a boca de aspiração. Insira quaisquer outros tubos (contendo agonistas, inibidores, etc) em sua posição apropriada ao longo da câmara. </li><li> Visualizar as células utilizando qualquer um, 1,4 objectiva 200X ou 400X abertura numérica e de um laser de árgon para excitar o corante Fluo-04:00 comprimento de onda de 488 nm (Figura 1A, parte inferior). As configurações do laser de potência deve ser ajustada de modo que o tubo fotomultiplicador (PMT) Não está saturado com a luz emitida. Além disso, a tensão através do PMT tem de ser optimizado para a detecção de luz máxima. </li><li> Sinais de&gt; 515 nm de emissão de passa-longo são coletados em velocidades de quadro de 2-5 seg / frame para a visualização de padrões oscilatórios lentos e 0,2-0,3 seg / quadro para a visualização rápida, ondas de cálcio globais (Figuras 3 e 4). </li><li> Depois de imagens estiver concluída, feche as seringas e remover toda a tubulação. Desmonte a câmara e repita os passos de 2,2-2,4. </li><li> Coletar dados como valores numéricos de intensidade de fluorescência ao longo do tempo e transferência à Imagem J (pacote de software fornecido como freeware pelo National Institutes of Health e podem ser encontradas em <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> ). </li><li> Veja as imagens de celular em tempo real e identificar regiões de interesse (ROI, ou seja, apical, basolateral, nuclear, etc) </li><li> Adquirir intensidades de fluorescência para estes ROIs erepresent como intensidade de fluorescência / linha de base da intensidade da fluorescência (isto é F/F0). </li><li> Gerar traçados traçando F / F 0 vs tempo. Além de traçados de imagens representativas são geralmente fornecidas e exibida usando pseudo. </li></ol> 3. Preparação de células pancreáticas acinares para Ensaios lesão celular <ol><li> Quentes DMEM F-12 mídia (sem vermelho de fenol) a 37 ° C. </li><li> Adicionar BSA (0,1% w / v final), e HCl (50 mM final) para os F-12 DMEM mídia. </li><li> Alíquotas de 50 ml de DMEM para um tubo cónico. </li><li> Preparar um tampão de colagenase por adição de 0,5 mg / ml (12 U / mL) de colagenase tipo IV de 10 ml de DMEM suplementado F-12 media. </li><li> Sacrificá um rato por asfixia CO 2. Orientar o animal em decúbito dorsal, e preparar a superfície abdominal por limpeza com álcool 70%. Realizar uma laparotomia para expor a cavidade abdominal. Dissecar o pâncreas e lave imediatamente em um pequeno barco pesando containing 6 ml de tampão de colagenase. </li><li> Picar o tecido com uma tesoura de dissecção multa para 3-5 minutos ou até que a solução resultante aparece uniformemente dispersas. </li><li> Transferir o tecido triturada para um balão de Erlenmeyer de 125 ml com 5 ml de tampão de colagenase adicional, em seguida, colocar num banho de água em ° C 37 ° C com agitação a 90 rpm durante 5 min. </li><li> Retire do agitador, para permitir que as células assentar brevemente, e troca com 5 ml de tampão de colagenase fresco. Em seguida incubar durante um adicional de 35 min num banho de água a 37 ° C com agitação a 90 rpm. </li><li> Vigorosamente ressuspender a digest, utilizando uma pipeta de transferência até a suspensão se torna homogénea, sem grumos de células visíveis. Em seguida, pipeta suavemente a suspensão de células através de nylon de malha pré-molhado num erlenmeyer. </li><li> Vigorosamente pipetar um adicional de 3 ml de DMEM fresco F-12, com meios (colagenase) através da malha, a fim de forçar através de quaisquer células restantes. </li><li> Adicionar outro 6-10 ml de DMEM F-12 mídia e umGUARDE as células que se contentar com 2-3 min. Repetir este passo duas vezes, e remover o sobrenadante, após a última lavagem. </li><li> Adicionar DMEM fresco F-12 media, ressuspender as células, e placa de 500 ul de suspensão celular em cada poço de uma placa de cultura de tecidos de 48 poços. As células devem assemelhar-se aos representados na Figura 1B. </li><li> Permitir que a placa para se sentar em um banho de água 37 ° C a 90 rpm por 5 min antes de adicionar agonistas. </li><li> Estimular as células com diferentes agonistas para 2-4 horas. </li><li> Inclinação da placa em um ângulo para permitir que as células que se contentar com cuidado e pipetar uma aliquota dos meios de comunicação (normalmente 100 l) e congelamento de flash em azoto líquido. </li><li> Flash congelar a suspensão de células remanescente. Congelamento de inflamação não é essencial para a medição de LDH. Como uma alternativa as amostras podem ser mantidas em gelo, se eles vão ser testadas no mesmo dia, ou armazenado em -20 ° C para armazenamento a longo prazo. </li></ol> 4. Medição de lactato desidrogenase Vazamento <ol><li&gt; Medida de lactato desidrogenase (LDH) essencialmente o vazamento utilizando os reagentes e as instruções fornecidas no kit de ensaio Cytotox 96 Non-Radioactive Cytotoxicity Promega (ver Tabela de reagentes e materiais específicos). </li><li> Reconstituir o substrato LDH (que é fornecido com o kit) com 12 ml de tampão de ensaio (também fornecidos). </li><li> Descongelar suspensão de células congeladas e amostras de meio num banho de água à temperatura ambiente. </li><li> Placa de 50 ul de cada amostra de material em uma placa de 96 poços. </li><li> Para a suspensão de células, adiciona o volume necessário de 10X de tampão de lise de modo que a concentração final é de 1X. Brevemente Vortex e incubar a 37 ° C durante 60 min. </li><li> Centrifugar as amostras lisadas a 250 xg durante 4 min a detritos sedimento celular. </li><li> Para lisados ​​celulares, placa 50 ul de uma diluição de 1:10 para cada poço de uma placa de 96 poços. </li><li> Adicionar 50 ul de substrato reconstituído a cada poço, incubar a placa no escuro à temperatura ambiente durante 30 min. </li><li> Parar a reacçãoadição de 50 ul da solução de paragem (fornecida com o kit) para cada poço. </li><li> Medir a absorvância a 490 nm. </li><li> Use a seguinte fórmula para calcular% LDH fugas. As densidades ópticas abaixo são corrigidos subtraindo uma leitura OD em branco de um poço contendo apenas PBS, substrato e solução de paragem. </li></ol> LDH em media = (Media OD492) x (fator de diluição media) x (vol. de mídia) * Uma vez que não é normalmente necessário para diluir amostras de mídia, o fator de diluição na maioria das vezes equal1. Total de LDH = ((Lysate OD 490) x (fator de diluição lysate) x (vol. em suspensão de células)) + ((Media OD 490) x (fator de diluição) x (vol. de mídia alíquota para amostragem)) <img alt="Equação 1" fo:src="/files/ftp_upload/50391/50391eq1highres.jpg" src="/files/ftp_upload/50391/50391eq1.jpg" /> 5. Infectando células pancreáticas acinares com Adenovirus <ol><li> Prepararcélulas acinares pancreáticas, como descrito na seção 3. </li><li> Seguindo o passo 3.12, adicionar 10 7 unidades infecciosas (IFUs) de adenovírus para a suspensão de células e incubar durante 30 min a 37 ° C. </li><li> Uniformemente placa de 2 ml de suspensão de células numa placa de 6 poços. </li><li> Para a maioria das construções de expressão, as células devem ser adequadamente infectado dentro de 18 horas, e para algumas construções luciferase, apenas dentro de 6 horas. </li></ol>

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O método de isolamento de células e ensaios subseqüentes descritos aqui representam poderosas ferramentas com as quais estudam os aspectos fisiológicos e fisiopatológicos do pâncreas exócrino. O método de isolamento de células pancreáticas acinares dispersas foi descrita pela primeira vez por Amesterdão e Jamieson, em 1972, 11. Os métodos aqui apresentados foram adaptados a partir de métodos de isolamento mais recentes descritos por Van Acker e colegas 12. Embora estas técnicas são a...

Estas alterações dinâmicas em cálcio intracelular são necessárias para os eventos de células acinares tanto fisiológicos e patológicos. Estes efeitos divergentes de cálcio são pensados ​​para resultar de padrões espaciais e temporais distintos de cálcio de sinalização 1. Por exemplo, a enzima e a secreção de fluido a partir da célula acinar estão ligadas a pontos do cálcio a partir de uma região limitada do pólo apical onde ocorre a secreção de 2. Em contraste, uma onda de cálcio global seguida de sinais de cálcio não-oscilatórias intensas está associada com eventos patológicos iniciais que conduzem a pancreatite aguda 3,4. Estes incluem a ativação intra-acinar protease, a secreção de enzimas reduzida e lesão celular acinar. O nosso laboratório utiliza isolado ácinos pancreáticos de estudar estes eventos patológicos iniciais que conduzem a doença, tanto in vivo como in vitro 5-7. Os métodos descritos aqui descrevem o isolamento de células acinares primárias com a finalidade de medir cyníveis de cálcio tosolic e lesão celular. Um método para a infecção adenoviral destas células também é fornecido.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Mice</td>
 <td>NCI</td>
 <td>N/A</td>
 <td>Male 20-30 grams; virtually any strain should yield comparable results.</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>American Bioanalytical</td>
 <td>AB00892</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>J.T. Baker</td>
 <td>3624-05</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>J.T. Baker</td>
 <td>3040-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Sigma</td>
 <td>M-8266</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride</td>
 <td>Fischer</td>
 <td>C79</td>
 <td></td>
 </tr>
 <tr>
 <td>Dextrose</td>
 <td>J.T. Baker</td>
 <td>1916-01</td>
 <td></td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Sigma</td>
 <td>G-8540</td>
 <td></td>
 </tr>
 <tr>
 <td>1X minimum Eagle's medium non-essential amino acid mixture</td>
 <td>Gibco</td>
 <td>11140-050</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Hydroxide</td>
 <td>EM</td>
 <td>SX0593</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>A7906</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Worthington</td>
 <td>4188</td>
 <td></td>
 </tr>
 <tr>
 <td>Soybean Trypsin Inhibitor</td>
 <td>Sigma</td>
 <td>T-9003</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbon Dioxide</td>
 <td>Matheson Gas</td>
 <td> 124-38-9 </td>
 <td></td>
 </tr>
 <tr>
 <td>125 ml Erlenmeyer plastic flask</td>
 <td>Crystalgen </td>
 <td>26-0005</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection kit</td>
 <td>Fine Science Tools</td>
 <td>14161-10</td>
 <td></td>
 </tr>
 <tr>
 <td>70% Ethanol</td>
 <td>LabChem</td>
 <td>LC222102</td>
 <td></td>
 </tr>
 <tr>
 <td>P1000, P100, P10 pipettes</td>
 <td>Gilson</td>
 <td>FA10005P</td>
 <td></td>
 </tr>
 <tr>
 <td>Weighing boat</td>
 <td>Heathrow Scientific</td>
 <td>HS1420A</td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic transfer pipettes</td>
 <td>USA Scientific</td>
 <td>1020-2500</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>352095</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td></td>
 </tr>
 <tr>
 <td>1.5 ml micro-centrifuge tube</td>
 <td>Fisher</td>
 <td>05-408-129</td>
 <td></td>
 </tr>
 <tr>
 <td>0.65 ml micro-centrifuge tube</td>
 <td>VWR</td>
 <td>20170-293</td>
 <td></td>
 </tr>
 <tr>
 <td>22 x 22 mm glass coverslips</td>
 <td>Fisher</td>
 <td>032811-9</td>
 <td></td>
 </tr>
 <tr>
 <td>Nitric Acid</td>
 <td>Fischer</td>
 <td>A483-212</td>
 <td></td>
 </tr>
 <tr>
 <td>Hydrochloric Acid</td>
 <td>Fischer</td>
 <td>A142-212</td>
 <td></td>
 </tr>
 <tr>
 <td>Deionized water</td>
 <td>N/A</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>18 x 18 mm coverslips</td>
 <td>Fischer</td>
 <td>021510-9</td>
 <td></td>
 </tr>
 <tr>
 <td>Laboratory film</td>
 <td>Parafilm</td>
 <td>PM-996</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-4AM</td>
 <td>Invitrogen</td>
 <td>F14201</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>Luer lock</td>
 <td>Becton Dickinson</td>
 <td>932777</td>
 <td></td>
 </tr>
 <tr>
 <td>60 ml syringe</td>
 <td>BD Bioscience</td>
 <td>DG567805</td>
 <td></td>
 </tr>
 <tr>
 <td>23 &frac34; gauge needle</td>
 <td>BD Bioscience</td>
 <td>9328270</td>
 <td></td>
 </tr>
 <tr>
 <td>PE50 tubing</td>
 <td>Clay Adam</td>
 <td>PE50-427411</td>
 <td></td>
 </tr>
 <tr>
 <td>Flat head screwdriver </td>
 <td>N/A</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM F-12, no Phenol Red</td>
 <td>Gibco</td>
 <td>21041-025 </td>
 <td></td>
 </tr>
 <tr>
 <td>30.5 gauge needle</td>
 <td>BD Bioscience</td>
 <td>305106</td>
 <td></td>
 </tr>
 <tr>
 <td>5 CC syringe</td>
 <td>BD Bioscience</td>
 <td>309603</td>
 <td></td>
 </tr>
 <tr>
 <td>25 ml Erlenmeyer flask</td>
 <td>Fischer</td>
 <td>FB50025</td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon mesh filter </td>
 <td>Nitex</td>
 <td>03-150/38</td>
 <td>150 &mu;m pore size</td>
 </tr>
 <tr>
 <td>48 well tissue culture plate</td>
 <td>Costar</td>
 <td>3548</td>
 <td></td>
 </tr>
 <tr>
 <td>96 well tissue culture plate </td>
 <td>Costar</td>
 <td>3795</td>
 <td></td>
 </tr>
 <tr>
 <td>6 well tissue culture plate</td>
 <td>Costar</td>
 <td>3506</td>
 <td></td>
 </tr>
 <tr>
 <td>Liquid nitrogen</td>
 <td>Matheson Gas</td>
 <td>7727-37-9</td>
 <td></td>
 </tr>
 <tr>
 <td>Cytotoxicity assay kit</td>
 <td>Promega</td>
 <td>G1782</td>
 <td></td>
 </tr>
 <tr>
 <td>Adeno-GFP</td>
 <td>N/A</td>
 <td>N/A</td>
 <td>Gift from J. Williams</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Ring stand with clamps</td>
 <td>United Scientific</td>
 <td>SET462</td>
 <td></td>
 </tr>
 <tr>
 <td>Perifusion chamber</td>
 <td>N/A</td>
 <td>N/A</td>
 <td>Designed by S.Z.H and colleagues at Yale University</td>
 </tr>
 <tr>
 <td>Vacuum line</td>
 <td>Manostat</td>
 <td>72-100-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Water bath with shaker</td>
 <td>Precision Scientific</td>
 <td>51220076</td>
 <td></td>
 </tr>
 <tr>
 <td>Confocal microscope</td>
 <td>Zeiss</td>
 <td>LSM 710</td>
 <td></td>
 </tr>
 <tr>
 <td>BioTek Synergy H1 plate reader</td>
 <td>BioTek</td>
 <td>11-120-534</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue culture hood</td>
 <td>Nuaire</td>
 <td>NU-425-600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue culture Incubator</td>
 <td>Thermo</td>
 <td>3110</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of pancreatic acinar cells for the purpose of calcium imaging, cell injury measurements, and adenoviral infection

As células acinares do pâncreas é a principal célula do parênquima do pâncreas exócrino e desempenha um papel primordial na secreção de enzimas pancreáticos no ducto pancreático. É também o local para o início da pancreatite. Aqui, nós descrevemos como células acinares são isolados a partir de sinais de cálcio intracelular tecido e pâncreas todo são medidos. Além disso, descrevem-se as técnicas de transfecção destas células com as construções adenovirais, e, subsequentemente, medindo a fuga de lactato desidrogenase, um marcador de lesão da célula, em condições que induzem a lesão das células acinares, in vitro. Estas técnicas proporcionam uma ferramenta poderosa para caracterizar acinar celular fisiologia e patologia.

Um exemplo de medições de cálcio de células acinares, em resposta a estímulos fisiológicos é fornecido na Figura 3. As células acinares foram carregados com o corante Fluo-cálcio 4 e perfundido com a acetilcolina análogo carbacol (CCh, 1 uM)) 8. Células reagiu, sob a forma de uma onda de cálcio, que tem início na região apical e propaga à região basolateral de 3,9. Traçados representativos mostrados na Figura 3B mostram o padrão típico de pico-pl...

Watch this Scientific Journal Video about Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection at JoVE.com

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

Nós descrevemos um método reprodutível de preparação de células acinares pancreáticas de rato de um rato com a finalidade de examinar os sinais de cálcio de células acinares e lesão celular com estímulos fisiologicamente e patologicamente relevantes. Um método para a infecção adenoviral destas células também é fornecido.

Preparação de células pancreáticas acinares para Fins de cálcio Imaging, celulares Medidas ferimento e infecção adenoviral

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

preparation-pancreatic-acinar-cells-for-purpose-calcium-imaging-cell

Research

JoVE Journal

Biology

13.2K visualizações.  Children's Hospital of Pittsburgh of UPMC. Nós descrevemos um método reprodutível de preparação de células acinares pancreáticas de rato de um rato com a finalidade de examinar os sinais de cálcio de células acinares e lesão celular com estímulos fisiologicamente e patologicamente relevantes. Um método para a infecção adenoviral destas células também é fornecido.

Vídeo: Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolamento e Cultura do mouse primárias células pancreáticas acinares

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Biologia

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

Imagens de células Lesão de Membrana e Processos Subcelulares envolvidas no reparo

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Imagens de células vivas de primários de ratos neonatos Cardiomyocytes Após Adenoviral e Transdução Lentivirus Usando confocal Spinning Disk Microscopia

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex&#160;vivo culture will represent the in&#160;vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Isolated pancreatic acini retain their in&#160;vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.

Avaliando a capacidade de secreção de pâncreas Acinar Células

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

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 ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Medições de esfíncter uretral Cystometric e externo em ratos acordados com cateter implantado e eletrodos permitindo medições repetidas

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Técnicas de micromanipulação, permitindo a análise da dinâmica morfogenética e rotatividade dos reguladores do citoesqueleto

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análise da função de células Beta utilizando imagens de cálcio de célula única resolução em ilhotas de Zebrafish

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...