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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 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).
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. Place the flask in a 37 °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.
10. 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.
11. Allow the remaining cells to settle, and exchange the media with fresh BSA incubation buffer.
12. 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).
13. Remove the BSA incubation buffer and replace with HEPES incubation buffer.
14. Prepare a 750 μM, 100X stock of Fluo-4AM in 10% DMSO.
15. 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 μl of this suspension onto each 22x22 mm coverslip.
16. 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.

2. Measuring Calcium in Pancreatic Acinar Cells

1. 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.
2. Use fine tweezers to carefully grasp from its edge one coverslip containing cell suspension. Tilt the coverslip 45°, 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).
3. 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.
4. 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.
5. Long-pass emission signals of > 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).
6. After imaging is complete, close the syringes and remove all tubing. Disassemble the chamber and repeat steps 2.2-2.4.
7. 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 http://rsb.info.nih.gov/ij/).
8. View real-time cellular images and identify regions of interest (ROIs; i.e. apical, basolateral, nuclear, etc.)
9. Acquire fluorescence intensities for these ROIs and represent as fluorescence intensity/baseline fluorescence intensity (i.e. F/F0).
10. Generate tracings by plotting F/F₀ vs. time. In addition to tracings, representative images are commonly provided and displayed using pseudocolor.

3. Preparation of Pancreatic Acinar Cells for Cell Injury Assays

1. Warm DMEM F-12 media (without phenol red) to 37 °C.
2. Add BSA (0.1% w/v final), and HCl (50 μM final) to the DMEM F-12 media.
3. Aliquot 50 ml of DMEM into a conical tube.
4. 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.
5. 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.
6. Mince the tissue using fine dissection scissors for 3-5 min or until the resulting solution appears evenly dispersed.
7. Transfer the minced tissue to a 125 ml Erlenmeyer flask with 5 ml of additional collagenase buffer, then place into a 37 °C water bath with shaking at 90 rpm for 5 min.
8. 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 °C water bath with shaking at 90 rpm.
9. 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.
10. 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.
11. 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.
12. Add fresh DMEM F-12 media, resuspend the cells, and plate 500 μl of cell suspension into each well of a 48-well tissue culture plate. The cells should resemble those depicted in Figure 1B.
13. Allow the plate to sit in a 37 °C water bath at 90 rpm for 5 min before adding agonists.
14. Stimulate the cells with varying agonists for 2-4 hr.
15. Tilt the plate at an angle to allow the cells to settle and carefully pipette out an aliquot of media (usually 100 μl) and flash freeze in liquid nitrogen.
16. 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 °C for long term storage.

4. Measuring Lactate Dehydrogenase Leakage

1. 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).
2. Reconstitute the LDH substrate (that is provided in the kit) with 12 ml of assay buffer (also provided).
3. Thaw frozen cell suspension and media samples in a water bath at room temperature.
4. Plate 50 μl of each media sample in a 96-well plate.
5. 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 °C for 60 min.
6. Centrifuge lysed samples at 250 x g for 4 min to pellet cell debris.
7. For cell lysates, plate 50 μl of a 1:10 dilution into each well of a 96 well plate.
8. Add 50 μl of reconstituted substrate to each well, incubate the plate in the dark at room temperature for 30 min.
9. Stop the reaction by adding 50 μl of the stop solution (provided in the kit) to each well.
10. Measure absorbance at 490 nm.
11. 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.

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))

5. Infecting Pancreatic Acinar Cells with Adenovirus

1. Prepare pancreatic acinar cells as described in section 3.
2. Following step 3.12, add 10⁷ infectious units (IFUs) of adenovirus to the cell suspension and incubate for 30 min at 37 °C.
3. Evenly plate 2 ml of cell suspension in a 6-well plate.
4. For most expression constructs, cells should be adequately infected within 18 hr; and for some luciferase constructs, just within 6 hr.

Results

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...

Discussion

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...

Materials

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