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Biology

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

Published: July 5th, 2013

DOI:

10.3791/50391

1Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Pittsburgh of UPMC, 2Department of Surgery, Tufts University Medical Center

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 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.

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 s....

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1. Preparing Pancreatic Acinar Cells for Calcium Imaging

  1. 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 × minimum Eagle's medium non-essential amino acids. Adjust the final solution to pH 7.4 with NaOH.
  2. Prepare a BSA incubation buffer by adding BSA (1% w/v final) to 25 ml of the HEPES incubation buffer (described above).
  3. Prepare a collagenase digestion buffer by.......

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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 μM)) 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.......

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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.......

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

....

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Name Company Catalog Number Comments
Name Company Catalogue Number Comments
Mice NCI N/A Male 20-30 grams; virtually any strain should yield comparable results.
HEPES American Bioanalytical AB00892
Sodium Chloride J.T. Baker 3624-05
Potassium Chloride J.T. Baker 3040-01
Magnesium Chloride Sigma M-8266
Calcium Chloride Fischer C79
Dextrose J.T. Baker 1916-01
L-Glutamine Sigma G-8540
1X minimum Eagle's medium non-essential amino acid mixture Gibco 11140-050
Sodium Hydroxide EM SX0593
Bovine Serum Albumin Sigma A7906
Collagenase Worthington 4188
Soybean Trypsin Inhibitor Sigma T-9003
Carbon Dioxide Matheson Gas 124-38-9
125 ml Erlenmeyer plastic flask Crystalgen 26-0005
Dissection kit Fine Science Tools 14161-10
70% Ethanol LabChem LC222102
P1000, P100, P10 pipettes Gilson FA10005P
Weighing boat Heathrow Scientific HS1420A
Plastic transfer pipettes USA Scientific 1020-2500
15 ml conical tubes BD Falcon 352095
50 ml conical tubes BD Falcon 352070
1.5 ml micro-centrifuge tube Fisher 05-408-129
0.65 ml micro-centrifuge tube VWR 20170-293
22 x 22 mm glass coverslips Fisher 032811-9
Nitric Acid Fischer A483-212
Hydrochloric Acid Fischer A142-212
Deionized water N/A N/A
18 x 18 mm coverslips Fischer 021510-9
Laboratory film Parafilm PM-996
Fluo-4AM Invitrogen F14201
Dimethylsulfoxide Sigma D2650
Luer lock Becton Dickinson 932777
60 ml syringe BD Bioscience DG567805
23 ¾ gauge needle BD Bioscience 9328270
PE50 tubing Clay Adam PE50-427411
Flat head screwdriver N/A N/A
DMEM F-12, no Phenol Red Gibco 21041-025
30.5 gauge needle BD Bioscience 305106
5 CC syringe BD Bioscience 309603
25 ml Erlenmeyer flask Fischer FB50025
Nylon mesh filter Nitex 03-150/38 150 μm pore size
48 well tissue culture plate Costar 3548
96 well tissue culture plate Costar 3795
6 well tissue culture plate Costar 3506
Liquid nitrogen Matheson Gas 7727-37-9
Cytotoxicity assay kit Promega G1782
Adeno-GFP N/A N/A Gift from J. Williams
Equipment
Ring stand with clamps United Scientific SET462
Perifusion chamber N/A N/A Designed by S.Z.H and colleagues at Yale University
Vacuum line Manostat 72-100-000
Water bath with shaker Precision Scientific 51220076
Confocal microscope Zeiss LSM 710
BioTek Synergy H1 plate reader BioTek 11-120-534
Tissue culture hood Nuaire NU-425-600
Tissue culture Incubator Thermo 3110

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