Questo lavoro è stato sostenuto da un National Institutes of Health di Grant DK083327 e DK093491 (a CSPS).

1. Preparazione acinose pancreatiche Cellule per Imaging di calcio <ol><li> Preparare HEPES tampone di incubazione contenente HEPES 20 mM, 95 mM NaCl, 4,7 mM KCl, 0,6 mM MgCl2, 1.3 mM CaCl 2, 10 mM di glucosio, 2 mM glutammina, e 1 × medie aminoacidi non essenziali del minimo Aquila. Regolare la soluzione finale a pH 7.4 con NaOH. </li><li> Preparare un tampone di incubazione aggiungendo BSA BSA (1% w / v finale) a 25 ml di tampone di incubazione HEPES (descritta sopra). </li><li> Preparare un tampone di digestione collagenasi aggiungendo 1,1 mg / ml (200 Unità / ml) di tipo-4 collagenasi e 1 mg / ml di inibitore della tripsina di soia per 6 ml di BSA tampone di incubazione (descritta sopra). </li><li> Acid-wash 22x22 mm coprioggetto immergendo in due parti HNO 3 e una parte di HCl per 2 ore. Poi decantare con acqua deionizzata e conservare in etanolo al 70%. </li><li> Euthanize un mouse da CO 2 asfissia. Orientare l&#39;animale in posizione supina, e preparare la superficie addominale da cleAning con il 70% di etanolo. Eseguire una laparotomia per esporre la cavità addominale. Sezionare il pancreas e sciacquare immediatamente in una piccola barca a pesare contenente 6 ml di tampone di digestione collagenasi. </li><li> Sezionare grossi pezzi di vasi sanguigni di grassi o visibile, quindi rimuovere 5 ml di tampone di digestione collagenasi e aggiungerlo di nuovo al tubo da 15 ml. </li><li> Utilizzando sottili forbici dissezione tritare il pancreas nella piccola barca pesa contenente 1 ml di tampone di digestione collagenasi. Tritare fino a quando la soluzione risultante viene uniformemente disperso. </li><li> Trasferire il prodotto macinate in un pallone di Erlenmeyer da 125 ml di plastica, e aggiungere il restante 5 ml di tampone di digestione collagenasi al contenitore. Assicurarsi che il tessuto è completamente sommerso nel buffer. </li><li> Porre il pallone in un bagno di 37 ° C acqua e agitare a 90 rpm per 30 min. Durante questo breve periodo di inattività, preparare agonisti attivazione di calcio (ad esempio caerulein, carbachol) e sciacquare il coperchio lavata con acido 22x22 mmscivola con acqua deionizzata. Secco, e poi posto sulla cima di una superficie piana rivestita da film di laboratorio. </li><li> Dopo 30 min la digest è completa, trasferire la sospensione posteriore al tubo da 15 ml e permettono alle cellule di stabilirsi. Pipetta con cautela il tampone di digestione collagenasi e sostituirlo con 6 ml di tampone di incubazione BSA. Agitare vigorosamente il tubo a mano per 10 sec per disperdere le cellule in cluster più piccole. Subito dopo l&#39;agitazione, rimuovere qualsiasi grande, detriti galleggianti da parte dei media instabili. </li><li> Lasciare le cellule rimanenti di stabilirsi, e scambiare i mezzi di comunicazione con il tampone di incubazione BSA fresca. </li><li> Ripetere il punto 1.11 due volte, fino a quando la sospensione è composta di soli piccoli cluster che sono solo finemente visibili ad occhio nudo. Le cellule dovrebbero assomigliare a quelli raffigurati nella Figura 1A (riga superiore). </li><li> Rimuovere il tampone di incubazione BSA e sostituirlo con HEPES tampone di incubazione. </li><li> Preparare una 750 micron, magazzino 100X di Fluo-04:00 nel 10% di DMSO. </li><li> Caricare le cellule nel tubo da 15 ml contenente la sospensione cellulare e HEPES BSA tampone privo di incubazione. Per fare questo, aggiungere un volume adeguato (diluizione 1:100) della soluzione di riserva alla sospensione cellulare. Poi piatto 500 ml di questa sospensione su ogni coprioggetto 22x22 mm. </li><li> Tenere coprioggetto al buio a temperatura ambiente. Prendere la prima a Ca 2 + misurazioni 30 min dopo il caricamento. Il colorante viene rimosso automaticamente dal mezzo all&#39;avviamento del perfusione, che dovrebbe avvenire 30 min dopo il caricamento colorante. </li></ol> 2. Misurazione di calcio in cellule acinose pancreatiche <ol><li> Sciacquare ogni perfusione set-up con acqua deionizzata e riempire con una quantità appropriata di tampone o agonista. Prime ogni siringa con il tampone o agonista per assicurare il flusso corretto. Bloccare siringhe a un anello riposare circa 1-2 metri sopra il palco microscopio. </li><li> Utilizzare una pinzetta sottile di cogliere con attenzione dal suo bordo un vetrino sospensione cellulare contenente. Inclinare il coperchioslip 45 °, e lasciare il tampone in eccesso di defluire. Posizionare il coprioggetto sopra della guarnizione di gomma con cellule sulla parte superiore del vetrino, e montare la camera (Figure 2A e 2B). </li><li> Fissare la camera di perfusione alla fase e inserire il tubo che contiene tampone nel primo ingresso della camera (Figura 2C). Ruotare la siringa contenente tampone e permettere che il buffer di perfusione su tutta la superficie del vetrino. Quando il buffer ha raggiunto l&#39;estremità opposta della camera, inserire una linea di vuoto dalla presa di vuoto. Inserire altri tubi (contenenti agonisti, inibitori, ecc) nella loro posizione appropriata lungo la camera. </li><li> Visualizzare le cellule utilizzando sia una, 1.4 Apertura obiettivo 200X o 400X numerico ed un laser ad argon per eccitare il colorante Fluo-04:00 alla lunghezza d&#39;onda di 488 nm (Figura 1A, in basso). Le impostazioni di potenza del laser devono essere regolati in modo che il tubo fotomoltiplicatore (PMT) Non è saturato dalla luce emessa. Inoltre, la tensione attraverso il PMT deve essere ottimizzato per il rilevamento massima luce. </li><li> Segnali di emissione a lungo passaggio di&gt; 515 nm sono raccolti a velocità fotogramma di 2-5 sec / frame per la visualizzazione di modelli di oscillatori lenti e 0,2-0,3 sec / frame per la visualizzazione più rapida, le onde di calcio globali (figure 3 e 4). </li><li> Dopo l&#39;imaging, chiudere le siringhe e rimuovere tutti i tubi. Smontare la camera e ripetere i passaggi 2,2-2,4. </li><li> Raccogliere i dati come valori numerici di intensità di fluorescenza nel tempo e trasferimento immagine J (pacchetto software fornito come freeware dal National Institutes of Health e può essere trovato a <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> ). </li><li> Guarda le immagini cellulari in tempo reale e di individuare le regioni di interesse (ROI, cioè apicale, basolaterale, nucleare, ecc) </li><li> Acquisire intensità di fluorescenza per queste zone di interesse e di represent come intensità di fluorescenza / basale intensità di fluorescenza (cioè F/F0). </li><li> Generare tracciati tracciando F / F 0 vs tempo. Oltre ai tracciati, immagini rappresentative sono comunemente forniti e visualizzati utilizzando pseudocolore. </li></ol> 3. Preparazione delle cellule acinose pancreatiche per saggi danno cellulare <ol><li> Caldo DMEM F-12 supporti (senza rosso fenolo) a 37 ° C. </li><li> Aggiungere BSA (0,1% w / v finale) e HCl (50 mM finale) ai DMEM F-12 media. </li><li> Aliquotare 50 ml di DMEM in un tubo conico. </li><li> Preparare un buffer collagenasi aggiungendo 0,5 mg / ml (12 U / ml) di collagenasi di tipo IV da 10 ml di DMEM integrato F-12 media. </li><li> Euthanize un mouse da CO 2 asfissia. Orientare l&#39;animale in posizione supina, e preparare la superficie addominale con la pulizia con il 70% di etanolo. Eseguire una laparotomia per esporre la cavità addominale. Sezionare il pancreas e sciacquare immediatamente in una piccola barca di peso containing 6 ml di tampone di collagenasi. </li><li> Tritare il tessuto utilizzando sottili forbici dissezione per 3-5 minuti o fino a quando la soluzione risultante appare uniformemente disperso. </li><li> Trasferire il tessuto macinata ad una beuta Erlenmeyer da 125 ml con 5 ml di tampone di collagenasi supplementare, poi posto in un bagno d&#39;acqua ° C 37 in agitazione a 90 rpm per 5 min. </li><li> Togliere dal shaker, consentono alle cellule di stabilirsi in breve, e lo scambio con 5 ml di tampone di collagenasi fresca. Poi incubare per un ulteriore 35 min in un bagno d&#39;acqua ° C 37 con agitazione a 90 rpm. </li><li> Vigorosamente risospendere il digest con una pipetta di trasferimento fino a quando la sospensione appare omogenea senza grumi di cellule evidenti. Poi pipettare delicatamente la sospensione cellulare attraverso una rete di nylon pre-bagnato in una beuta. </li><li> Vigorosamente Pipettare 3 ml di DMEM fresco aggiuntiva F-12 supporti (con collagenasi) attraverso la maglia, in modo da forzare attraverso qualsiasi celle rimanenti. </li><li> Aggiungere un altro 6-10 ml di DMEM F-12 media e unseguite la cellule per 2-3 min. Ripetere questa operazione due volte, e rimuovere il surnatante dopo il lavaggio finale. </li><li> Aggiungere DMEM fresco F-12 supporti, risospendere le cellule, e la piastra 500 microlitri di sospensione cellulare in ciascun pozzetto di una piastra di coltura tissutale a 48 pozzetti. Le celle dovrebbero somigliare quelli raffigurati in Figura 1B. </li><li> Lasciare che la piastra di sedersi in un 37 ° C a bagnomaria a 90 rpm per 5 minuti prima di aggiungere agonisti. </li><li> Stimolare le cellule con diversi agonisti per 2-4 ore. </li><li> Inclinare la piastra con un angolo per permettere alle cellule di stabilirsi e Pipettare attentamente fuori una aliquota di mezzi (in genere 100 ml) e flash congelare in azoto liquido. </li><li> Flash congelare la sospensione cellulare rimanente. Flash congelamento non è essenziale per le misure di LDH. In qualità di campioni alternativi possono essere tenuti in ghiaccio se saranno analizzati il ​​giorno stesso o conservati in -20 ° C per la conservazione a lungo termine. </li></ol> 4. Misurare lattato deidrogenasi di dispersione <ol><li&gt; Misura di lattato deidrogenasi (LDH) perdite principalmente utilizzando i reagenti e le istruzioni fornite nel Promega Cytotox 96 non radioattivo citotossicità kit di analisi (vedi Tabella di reagenti e materiali specifici). </li><li> Ricostituire il substrato LDH (fornito nel kit) con 12 ml di tampone di saggio (anche fornito). </li><li> Scongelare sospensione cellulare campioni congelati e multimediali in un bagno di acqua a temperatura ambiente. </li><li> Tavola 50 microlitri di ciascun campione media in una piastra a 96 pozzetti. </li><li> Alla sospensione cellulare, aggiungere il volume necessario di tampone di lisi 10X in modo che la concentrazione finale è 1X. Brevemente nel vortex e incubare a 37 ° C per 60 min. </li><li> Centrifugare i campioni lisati a 250 xg per 4 min a detriti cellulari pellet. </li><li> Per lisati cellulari, piastra 50 pl di una diluizione 1:10 in ciascun pozzetto di una piastra a 96 pozzetti. </li><li> Aggiungere 50 ml di substrato ricostituito in ogni pozzetto, incubare la piastra al buio a temperatura ambiente per 30 min. </li><li> Bloccare la reazioneaggiungendo 50 microlitri della soluzione di arresto (fornito nel kit) a ciascun pozzetto. </li><li> Misurare l&#39;assorbanza a 490 nm. </li><li> Utilizzare le seguenti formule per calcolare% di dispersione LDH. Le densità ottiche inferiori vengono corrette sottraendo un OD lettura vuota da un pozzo contenente solo PBS, substrato e soluzione di bloccaggio. </li></ol> LDH nei media = (Media OD492) x (fattore di diluizione media) x (vol. di media) * Dal momento che è di solito necessario per diluire i campioni dei media, il fattore di diluizione sarà molto spesso equal1. Totale LDH = ((lisato OD 490) x (lisato fattore di diluizione) x (vol. in sospensione cellulare)) + ((Media OD 490) x (fattore di diluizione) x (vol. di supporti aliquotati per il campionamento)) <img alt="Equazione 1" fo:src="/files/ftp_upload/50391/50391eq1highres.jpg" src="/files/ftp_upload/50391/50391eq1.jpg" /> 5. Infettare cellule acinose pancreatiche con Adenovirus <ol><li> Prepararecellule acinose pancreatiche come descritto nella sezione 3. </li><li> Seguendo passo 3.12, aggiungere 10 7 unità infettive (IFUS) di adenovirus alla sospensione cellulare e incubare per 30 min a 37 ° C. </li><li> Uniformemente piastra 2 ml di sospensione cellulare in una piastra da 6 pozzetti. </li><li> Per la maggior parte dei costrutti di espressione, le cellule devono essere adeguatamente infettati entro 18 ore, e per alcuni costrutti luciferasi, appena entro 6 ore. </li></ol>

<ol>
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Il metodo di isolamento delle cellule e saggi successivi raffigurati qui rappresentano potenti strumenti con cui studiare le caratteristiche fisiologiche e fisiopatologiche del pancreas esocrino. Il metodo per isolare le cellule acinose pancreatiche dispersi è stato descritto da Amsterdam e Jamieson nel 1972 11. I metodi qui presentati sono stati adattati da più recenti metodi di isolamento descritte da Van Acker e colleghi 12. Sebbene queste tecniche sono altamente riproducibili e facilmente app...

Cambiamenti dinamici nel calcio citosolico sono necessari per gli eventi delle cellule acinose sia fisiologiche che patologiche. Questi effetti divergenti di calcio sono pensati per derivare da diversi modelli spaziali e temporali di calcio di segnalazione 1. Per esempio, enzimi e la secrezione fluido dalla cellule acinose sono legate a picchi di calcio da una regione ristretta del polo apicale in cui la secrezione avviene 2. Al contrario, un&#39;onda calcio globale seguito da intensi segnali di calcio non-oscillatorie è associato ad eventi patologici precoci che portano alla pancreatite acuta 3,4. Questi includono attivazione intra-acinare proteasi, enzima secrezione ridotta e danno cellulare acinare. Il nostro laboratorio utilizza isolato acini pancreatici per studiare questi eventi patologici precoci che portano alla malattia, sia in vivo che in vitro 5-7. I metodi qui descritti descrivono isolamento di cellule acinose primarie a scopo di misurare cylivelli di calcio tosolic e lesioni delle cellule. Viene inoltre fornito un metodo per l&#39;infezione adenovirale di queste cellule.

<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

L&#39;cellule acinose pancreatiche è il principale parenchima cellulare del pancreas esocrino e svolge un ruolo primario nella secrezione di enzimi pancreatici nel dotto pancreatico. E &#39;anche il sito per l&#39;avvio di pancreatite. Qui si descrive come le cellule acinose sono isolati da tutto il tessuto del pancreas e dei segnali intracellulari di calcio sono misurati. Inoltre, si descrivono le tecniche di trasfezione queste cellule con costrutti adenovirali, e successivamente misurando la fuoriuscita di lattato deidrogenasi, un marker di danno cellulare, in condizioni che inducono lesioni cellule acinose in vitro. Queste tecniche forniscono un potente strumento per la caratterizzazione delle cellule acinose fisiologia e patologia.

Un esempio di misurazioni calcio delle cellule acinose in risposta a stimoli fisiologici è fornita in figura 3. Cellule acinose state caricate con il colorante calcio Fluo-4 e perfusi con l&#39;acetilcolina analogico carbachol (CCH, 1 mM)) 8. Cellule risposto nella forma di un&#39;onda di calcio che avvia nella regione apicale e propaga alla regione basolaterale 3,9. Tracciati Rappresentante mostrato in Figura 3B dimostrano il tipico modello di punta plateau comun...

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

Descriviamo un metodo riproducibile di preparazione del mouse pancreatiche cellule acinose da un mouse con lo scopo di esaminare i segnali di calcio delle cellule acinose e lesioni cellulari con stimoli fisiologicamente e patologicamente rilevanti. Viene inoltre fornito un metodo per l&#39;infezione adenovirale di queste cellule.

Preparazione delle cellule acinose pancreatiche per lo Scopo di Imaging di calcio, cellulari lesioni Misure e Infezione 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 Views.  Children's Hospital of Pittsburgh of UPMC. Descriviamo un metodo riproducibile di preparazione del mouse pancreatiche cellule acinose da un mouse con lo scopo di esaminare i segnali di calcio delle cellule acinose e lesioni cellulari con stimoli fisiologicamente e patologicamente rilevanti. Viene inoltre fornito un metodo per l'infezione adenovirale di queste cellule.

Video: 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 del mouse principale acinose pancreatiche Cellule

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 ...

Ricerca

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.

Imaging Membrana cellulare Pregiudizio e processi subcellulari coinvolti nella riparazione

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.

Imaging cellulare dal vivo di primaria ratto neonato Cardiomyocytes seguito Adenoviral e lentivirali trasduzione Uso confocale 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.

Valutare la capacità secretoria delle cellule pancreatiche acinari

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.

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

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.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

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

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.

Cistometrici ed esterni misure dello sfintere uretrale in ratti svegli con catetere impiantato ed elettrodi consentendo misurazioni ripetute

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.

Tecniche di micromanipolazione che consente analisi delle dinamiche morfogenetiche e fatturato dei regolatori del citoscheletro

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.

Analisi della funzione Beta-cellulare usando la formazione immagine del calcio cella singola risoluzione in Zebrafish isolotti

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.

Imaging calcio Dynamics in sottopopolazioni di cellule di islet pancreatica del topo

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