This work was supported by a National Institutes of Health Grant DK083327, and DK093491 (to S.Z.H.).

1. Preparing Pancreatic Acinar Cells for Calcium Imaging
<ol>
	<li>Prepare HEPES incubation buffer containing 20 mM HEPES, 95 mM NaCl, 4.7 mM KCl, 0.6 mM MgCl2, 1.3 mM CaCl2, 10 mM glucose, 2 mM glutamine, and 1 &times; minimum Eagle's medium non-essential amino acids. Adjust the final solution to pH 7.4 with NaOH.</li>
	<li>Prepare a BSA incubation buffer by adding BSA (1% w/v final) to 25 ml of the HEPES incubation buffer (described above).</li>
	<li>Prepare a collagenase digestion buffer by adding 1.1 mg/ml (200 Units/ml) type-4 collagenase and 1 mg/ml soybean trypsin inhibitor to 6 ml of BSA incubation buffer (described above).</li>
	<li>Acid-wash 22x22 mm coverslips by immersing in two parts HNO3 and one part HCl for 2 hr. Then decant using DI water and store in 70% ethanol.</li>
	<li>Euthanize one mouse by CO2 asphyxiation. Orient the animal in the supine position, and prepare the abdominal surface by cleaning with 70% ethanol. Perform a laparotomy to expose the abdominal cavity. Dissect out the pancreas and immediately rinse in a small weigh boat containing 6 ml of collagenase digestion buffer.</li>
	<li>Dissect away any large pieces of fat or visible blood vessels, then remove 5 ml of collagenase digestion buffer and add it back to the 15 ml conical tube.</li>
	<li>Using fine dissection scissors mince the pancreas in the small weighing boat containing 1 ml of collagenase digestion buffer. Mince until the resulting solution appears evenly dispersed.</li>
	<li>Transfer the minced product to a 125 ml Erlenmeyer plastic flask, and add the remaining 5 ml of collagenase digestion buffer to the container. Make sure the tissue is fully submerged in buffer.</li>
	<li>Place the flask in a 37 &deg;C water bath and shake at 90 rpm for 30 min. During this short period of downtime, prepare calcium activating agonists (e.g. caerulein, carbachol) and rinse the 22x22 mm acid-washed coverslips with DI water. Dry, and then place on top of a flat surface lined by laboratory film.</li>
	<li>After the 30 min digest is complete, transfer the suspension back to the 15 ml conical tube and allow the cells to settle. Carefully pipette out the collagenase digestion buffer and replace with 6 ml of BSA incubation buffer. Vigorously shake the tube by hand for 10 sec in order to disperse the cells into smaller clusters. Immediately upon shaking, remove any large, floating debris from the unsettled media.</li>
	<li>Allow the remaining cells to settle, and exchange the media with fresh BSA incubation buffer.</li>
	<li>Repeat step 1.11 twice, until the suspension is comprised of only small clusters which are only finely visible to the naked eye. The cells should resemble those depicted in Figure 1A (top row).</li>
	<li>Remove the BSA incubation buffer and replace with HEPES incubation buffer.</li>
	<li>Prepare a 750 &mu;M, 100X stock of Fluo-4AM in 10% DMSO.</li>
	<li>Load the cells in the 15 ml conical tube containing the cell suspension and BSA-free HEPES incubation buffer. To do this, add an appropriate volume (1:100 dilution) of the stock solution to the cell suspension. Then plate 500 &mu;l of this suspension onto each 22x22 mm coverslip.</li>
	<li>Keep coverslips in the dark at room temperature. Take the first Ca2+ measurements 30 min after loading. The dye is automatically removed from the medium upon starting the perfusion, which should occur 30 min after dye loading.</li>
</ol>
2. Measuring Calcium in Pancreatic Acinar Cells
<ol>
	<li>Rinse each perfusion set-up with DI water and fill with an appropriate amount of buffer or agonist. Prime each syringe with buffer or agonist to insure proper flow. Clamp syringes to a ring stand approximately 1-2 feet above the microscope stage.</li>
	<li>Use fine tweezers to carefully grasp from its edge one coverslip containing cell suspension. Tilt the coverslip 45&deg;, and allow the excess buffer to run off. Place the coverslip on top of the rubber gasket with the cells on top of the coverslip, and assemble the chamber (Figures 2A and 2B).</li>
	<li>Secure the perfusion chamber to the stage and insert the tubing which contains buffer into the first inlet of the chamber (Figure 2C). Turn the syringe containing buffer on and allow the buffer to perfuse over the entire surface of the coverslip. Once the buffer has reached the opposite end of the chamber, insert a vacuum line into the vacuum inlet. Insert any other tubes (containing agonist, inhibitors, etc.) into their appropriate position along the chamber.</li>
	<li>Visualize the cells using either a 200X or 400X, 1.4 numerical aperture objective and an argon laser to excite the Fluo-4AM dye at a wavelength of 488 nm (Figure 1A, bottom). The power settings of the laser should be adjusted such that the photomultiplier tube (PMT) is not saturated by the emitted light. In addition, the voltage across the PMT needs to be optimized for maximum light detection.</li>
	<li>Long-pass emission signals of &gt; 515 nm are collected at frame speeds of 2-5 sec/frame for visualizing slow oscillatory patterns and 0.2-0.3 sec/frame for visualizing faster, global calcium waves (Figures 3 and 4).</li>
	<li>After imaging is complete, close the syringes and remove all tubing. Disassemble the chamber and repeat steps 2.2-2.4.</li>
	<li>Collect data as numerical values of fluorescence intensity over time and transfer to Image J (software package provided as freeware by the National Institutes of Health and can be found at <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>).</li>
	<li>View real-time cellular images and identify regions of interest (ROIs; i.e. apical, basolateral, nuclear, etc.)</li>
	<li>Acquire fluorescence intensities for these ROIs and represent as fluorescence intensity/baseline fluorescence intensity (i.e. F/F0).</li>
	<li>Generate tracings by plotting F/F0 vs. time. In addition to tracings, representative images are commonly provided and displayed using pseudocolor.</li>
</ol>
3. Preparation of Pancreatic Acinar Cells for Cell Injury Assays
<ol>
	<li>Warm DMEM F-12 media (without phenol red) to 37 &deg;C.</li>
	<li>Add BSA (0.1% w/v final), and HCl (50 &mu;M final) to the DMEM F-12 media.</li>
	<li>Aliquot 50 ml of DMEM into a conical tube.</li>
	<li>Prepare a collagenase buffer by adding 0.5 mg/ml (12 U/ml) of collagenase type IV to 10 ml of supplemented DMEM F-12 media.</li>
	<li>Euthanize one mouse by CO2 asphyxiation. Orient the animal in the supine position, and prepare the abdominal surface by cleaning with 70% ethanol. Perform a laparotomy to expose the abdominal cavity. Dissect out the pancreas and immediately rinse in a small weighing boat containing 6 ml of collagenase buffer.</li>
	<li>Mince the tissue using fine dissection scissors for 3-5 min or until the resulting solution appears evenly dispersed.</li>
	<li>Transfer the minced tissue to a 125 ml Erlenmeyer flask with 5 ml of additional collagenase buffer, then place into a 37 &deg;C water bath with shaking at 90 rpm for 5 min.</li>
	<li>Remove from the shaker, allow cells to briefly settle, and exchange with 5 ml of fresh collagenase buffer. Then incubate for an additional 35 min in a 37 &deg;C water bath with shaking at 90 rpm.</li>
	<li>Vigorously resuspend the digest using a transfer pipette until the suspension appears homogeneous with no obvious cell clumps. Then gently pipette the cell suspension through a pre-wet nylon mesh into a conical flask.</li>
	<li>Vigorously pipette an additional 3 ml of fresh DMEM F-12 media (with collagenase) through the mesh, in order to force through any remaining cells.</li>
	<li>Add another 6-10 ml of DMEM F-12 media and allow the cells to settle for 2-3 min. Repeat this step twice, and remove the supernatant after the final wash.</li>
	<li>Add fresh DMEM F-12 media, resuspend the cells, and plate 500 &mu;l of cell suspension into each well of a 48-well tissue culture plate. The cells should resemble those depicted in Figure 1B.</li>
	<li>Allow the plate to sit in a 37 &deg;C water bath at 90 rpm for 5 min before adding agonists.</li>
	<li>Stimulate the cells with varying agonists for 2-4 hr.</li>
	<li>Tilt the plate at an angle to allow the cells to settle and carefully pipette out an aliquot of media (usually 100 &mu;l) and flash freeze in liquid nitrogen.</li>
	<li>Flash freeze the remaining cell suspension. Flash freezing is not essential for LDH measurements. As an alternative samples can be kept on ice if they will be assayed the same day or stored in -20 &deg;C for long term storage.</li>
</ol>
4. Measuring Lactate Dehydrogenase Leakage
<ol>
	<li>Measure lactate dehydrogenase (LDH) leakage primarily using the reagents and instructions supplied in the Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (see Table of specific reagents and materials).</li>
	<li>Reconstitute the LDH substrate (that is provided in the kit) with 12 ml of assay buffer (also provided).</li>
	<li>Thaw frozen cell suspension and media samples in a water bath at room temperature.</li>
	<li>Plate 50 &mu;l of each media sample in a 96-well plate.</li>
	<li>To the cell suspension, add the necessary volume of 10X lysis buffer so that the final concentration is 1X. Vortex briefly and incubate at 37 &deg;C for 60 min.</li>
	<li>Centrifuge lysed samples at 250 x g for 4 min to pellet cell debris.</li>
	<li>For cell lysates, plate 50 &mu;l of a 1:10 dilution into each well of a 96 well plate.</li>
	<li>Add 50 &mu;l of reconstituted substrate to each well, incubate the plate in the dark at room temperature for 30 min.</li>
	<li>Stop the reaction by adding 50 &mu;l of the stop solution (provided in the kit) to each well.</li>
	<li>Measure absorbance at 490 nm.</li>
	<li>Use the following formulae to calculate %LDH leakage. The optical densities below are corrected by subtracting out a blank OD reading from a well containing only PBS, substrate, and stop solution.</li>
</ol>
LDH in media = (Media OD492) x (media dilution factor) x (vol. of media)*Since it is usually unnecessary to dilute media samples, the dilution factor will most often equal1.
Total LDH = ((Lysate OD490) x (lysate dilution factor) x (vol. in cell suspension))+ ((Media OD490) x (dilution factor) x (vol. of media aliquoted for sampling))
<img alt="Equation 1" fo:src="/files/ftp_upload/50391/50391eq1highres.jpg" src="/files/ftp_upload/50391/50391eq1.jpg" />
5. Infecting Pancreatic Acinar Cells with Adenovirus
<ol>
	<li>Prepare pancreatic acinar cells as described in section 3.</li>
	<li>Following step 3.12, add 107 infectious units (IFUs) of adenovirus to the cell suspension and incubate for 30 min at 37 &deg;C.</li>
	<li>Evenly plate 2 ml of cell suspension in a 6-well plate.</li>
	<li>For most expression constructs, cells should be adequately infected within 18 hr; and for some luciferase constructs, just within 6 hr.</li>
</ol>

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	<li>Toescu, E. C., Lawrie, A. M., Petersen, O. H., Gallacher, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+distribution+of+agonist-evoked+cytoplasmic+Ca2++signals+in+exocrine+acinar+cells+analysed+by+digital+image+microscopy.">Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy.</a> Embo J. 11, 1623-1629 (1992).</li><li>Ito, K., Miyashita, Y., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolar+and+submicromolar+Ca2++spikes+regulating+distinct+cellular+functions+in+pancreatic+acinar+cells.">Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells.</a> Embo J. 16, 242-251 (1997).</li><li>Husain, S. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ryanodine+receptor+mediates+early+zymogen+activation+in+pancreatitis.">The ryanodine receptor mediates early zymogen activation in pancreatitis.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 14386-14391 (2005).</li><li>Raraty, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+enzyme+activation+and+vacuole+formation+in+the+apical+granular+region+of+pancreatic+acinar+cells.">Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 13126-13131 (2000).</li><li>Orabi, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dantrolene+mitigates+caerulein-induced+pancreatitis+in+vivo+in+mice.">Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 299, G196-G204 (2009).</li><li>Shah, A. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease+Activation+during+in+vivo+Pancreatitis+is+Dependent+upon+Calcineurin+Activation.">Protease Activation during in vivo Pancreatitis is Dependent upon Calcineurin Activation.</a> Am. J. Physiol. Gastrointest. Liver Physiol. , (2009).</li><li>Muili, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+and+genetic+inhibition+of+calcineurin+protects+against+carbachol-induced+pathologic+zymogen+activation+and+acinar+cell+injury.">Pharmacologic and genetic inhibition of calcineurin protects against carbachol-induced pathologic zymogen activation and acinar cell injury.</a> Am. J. Physiol. Gastrointest. Liver Physiol. , (2012).</li><li>Orabi, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+enhances+carbachol-induced+protease+activation+and+accelerates+Ca2++waves+in+isolated+rat+pancreatic+acini.">Ethanol enhances carbachol-induced protease activation and accelerates Ca2+ waves in isolated rat pancreatic acini.</a> J. Biol. Chem. 286, 14090-14097 (2011).</li><li>Nathanson, M. H., Padfield, P. J., O'Sullivan, A. J., Burgstahler, A. D., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Ca2++wave+propagation+in+pancreatic+acinar+cells.">Mechanism of Ca2+ wave propagation in pancreatic acinar cells.</a> J. Biol. Chem. 267, 18118-18121 (1992).</li><li>Reed, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+extracellular+pH+induces+damage+in+the+pancreatic+acinar+cell+by+enhancing+calcium+signaling.">Low extracellular pH induces damage in the pancreatic acinar cell by enhancing calcium signaling.</a> J. Biol. Chem. 286, 1919-1926 (2011).</li><li>Amsterdam, A., Jamieson, J. 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No conflict of interest declared.

The cell isolation method and subsequent assays depicted here represent powerful tools with which to study the physiological and pathophysiological features of the exocrine pancreas. The method for isolating dispersed pancreatic acinar cells was first described by Amsterdam and Jamieson in 1972 11. The methods presented here have been adapted from more recent isolation methods described by Van Acker and colleagues 12. Although these techniques are highly reproducible and easily learned, there...

Dynamic changes in cytosolic calcium are necessary for both physiological and pathological acinar cell events. These divergent effects of calcium are thought to result from distinct spatial and temporal patterns of calcium signaling 1. For example, enzyme and fluid secretion from the acinar cell are linked to calcium spikes from a restricted region of the apical pole where secretion takes place 2. In contrast, a global calcium wave followed by intense non-oscillatory calcium signals is associated with early pathological events which lead to acute pancreatitis 3,4. These include intra-acinar protease activation, reduced enzyme secretion, and acinar cell injury. Our lab uses isolated pancreatic acini to study these early pathological events which lead to disease both in vivo and in vitro 5-7. The methods detailed here describe isolation of primary acinar cells for the purpose of measuring cytosolic calcium levels and cell injury. A method for adenoviral infection of these cells is also provided.

<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

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 the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

An example of acinar cell calcium measurements in response to physiologic stimuli is provided in Figure 3. Acinar cells were loaded with the calcium dye Fluo-4 and perfused with the acetylcholine analogue carbachol (CCh; 1 &mu;M)) 8. Cells responded in the form of a calcium wave which initiates in the apical region and propagates to the basolateral region 3,9. Representative tracings shown in Figure 3B demonstrate the typical peak-plateau pattern commonly observed w...

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

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

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. We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

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

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Cancer Research

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Neuroscience

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.

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

Inducing Pancreatic Injury in Mice Through Intraperitoneal Drug Injection

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 &#181;g/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis (SAP).

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological ...

Immunology and Infection

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Medicine

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.

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

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Slicing Human Pancreas for Islet Function Research

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

Author Spotlight: Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the ...

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Source: Fleming Martinez, A. K. et. al. <a href="https://www.jove.com/v/59096/" target="_blank">Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture</a>. J. Vis. Exp. (2019).
This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

Source: Fleming Martinez, A. K. et. al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Pancreatic Cancer

Cell isolation & culture

Imaging Calcium Dynamics in Pancreatic Cells: A Technique to Study Real-time Changes in Cytosolic Calcium Concentration in Pancreatic Islet

Source: Hamilton, A., et. al. <a href="https://www.jove.com/v/59491/" target="_blank">Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells</a>. J. Vis. Exp. (2019).
This video describes the technique of imaging islet cells in real-time, to study the changes in cytosolic calcium concentration in the minor subpopulations of the pancreatic islet cells. 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. This can provide an insight into the cross talk between the blood glucose and the islet secretory output.

Source: Hamilton, A., et. al. Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells. J. Vis. Exp. (2019).
This video describes the ...