Isolation and Preparation of Acinar Cells for Atomic Force Microscopy.
Pancreatic acinar cells and hemi-acini were isolated using a minor modification of our published procedure (24). For each experiment, a male Sprague Dawley rat weighing 80-100 g was euthanized by ether inhalation. The pancreas was then dissected out and chopped into 0.5-mm3 pieces, which were mildly agitated for 15 min at 37°C in a siliconized glass tube with 5 ml of oxygenated buffer A (98 mM NaCl/4.8 mM KCl/2 mM CaCl2/1.2 mM MgCl2/0.1% bovine serum albumin/0.01% soybean trypsin inhibitor/25 mM Hepes, pH 7.4) containing 1000 units of collagenase. The suspension of acini was filtered through a 224-µm Spectra-Mesh (Spectrum Laboratory Products) polyethylene filter to remove large clumps of acini and undissociated tissue. The acini were washed six times, 50 ml per wash, with ice-cold buffer A. Isolated rat pancreatic acini and acinar cells were plated on Cell-Tak-coated (Collaborative Biomedical Products) glass coverslips. Two to three hours after plating, cells were imaged by the atomic force microscope before and during stimulation of secretion. Isolated acinar cells and small acinar preparations were used in the study because fusions of regulated secretory vesicles with the plasma membrane in pancreatic acini are confined to the apical region and are impossible to image by the atomic force microscope in whole tissue or large acinar preparations. Additionally, the secretagogue Mas7 has immediate and uniform access to all the acinar cells.
Isolated Pancreatic Acini for Light and Electron Microscopy.
After isolation of the rat pancreatic acini and acinar cells, they were resuspended in oxygenated buffer A and placed on a Cell-Tak-coated glass slide. Cells were observed and photographed using an Olympus 8MAX microscope. For electron microscopy, purified acinar cells in buffer A were centrifuged at 2000 × g for 2 min and the acinar pellet was resuspended in fixative. Cells were fixed in 4% buffered formaldehyde (PFA) for 2 h and in 8% PFA overnight, and the pellets were then embedded in 10% aqueous gelatin, infiltrated in 2.3 M sucrose containing 20% poly(vinylpyrrolidone), frozen by immersion in liquid nitrogen, and processed for cryosectioning (30). After the frozen block was sectioned at 
140°C and the sections were transferred to coated specimen grids, they were dried in the presence of uranyl acetate and methyl cellulose and examined in a Philips 410 transmission electron microscope operating at 80 kV.
Atomic Force Microscopy.
The atomic force microscope is a powerful instrument to observe and obtain three-dimensional images at the nanometer resolution range of living cells. Several studies using the atomic force microscope have previously been carried out on living cells. In the BAFM, a fine silicon or silicon nitride tip scans the surface of a sample. Any deflection of the tip due to surface topology is recorded. The BAFM reconstructs an image of the surface from the x, y, and z scan data, to develop a three-dimensional topology of any surface at the nanometer level. Cells attached to a Cell-Tak-coated glass coverslip were placed in a thermally controlled fluid chamber that allowed both rapid fluid exchange and the direct visualization of the living cells by an inverted microscope. The newly designed BAFM (Digital Instruments, Santa Barbara, CA) was used in conjunction with an inverted optical microscope (Olympus IX70). Images of the plasma membrane in these cells were obtained by the BAFM, working in the "contact" mode and using a very low vertical imaging force from imaging in the contact mode for biological material has been described elsewhere (31, 32). Silicon and silicon nitride tips were used with spring constants of 0.25 and 0.06 N·m
1, respectively. To determine the effect of force on the plasma membrane topology, control experiments where performed, where a scanning force of up to 10 nN over a 60-min period demonstrated no significant changes at the plasma membrane.
Atomic Force Imaging of Acinar Cells Following Exposure to Mas7 or Cytochalasin B.
For performing atomic force microscopy on stimulated cells, acinar cells were scanned for 10-60 min by the BAFM cantilever prior to Mas7 stimulation of secretion. Pancreatic acinar cells were then stimulated to secrete bu using 20 µM Mas7 (20 µl of a 2 mM stock of Mas7 was added to 2 ml of the buffer A incubation mixture). No significant thermal effects following the 20-µl additions to the fluid chamber were observed. Similarly, cells were scanned following a 30-min exposure to 20 µM cytochalasin B. Twenty microliters of a 2 mM stock of cytochalasin B was added to 2 ml of the buffer A incubation mixture at room temperature.
Amylase Measurement Following Exposure of Acinar Cells to Mas7 or Cytochalasin B.
Exocytosis from acinar cells was measured by determining the percentage of total cellular amylase release, following exposure of cells to a secretagogue or cytochalasin B. Amylase, one of the major contents of ZGs, was measured by the procedure of Bernfeld (33). In a typical amylase assay, rat pancreatic acini dissociated as single cells and clumps of 2-6 cells were used. Fifty to 75 cells in 200 µl of total reaction mixture (buffer A) in the presence or absence of Mas7 or cytochalasin B were incubated at room temperature. Following incubation, the cells were centrifuged at 2000 × g for 2 min in an Eppendorf microcentrifuge. The supernatant containing the secreted amylase was assayed. The cells in the remaining 100 µl of incubation mixture were sonicated, and the sonicate was diluted and assayed for amylase. From the above measurements, the total cellular amylase and percent release from the cells were calculated. Five microliters of the supernatant or lysed cell fractions was added to 95 µl of ice-cold amylase assay buffer (10 mM NaH2PO4/10 mM Na2HPO4/20 mM NaCl) placed in 12 × 75 mm glass tubes in an ice bath. The reaction was started by adding 100 µl of a 10-mg/ml potato starch amylase assay buffer solution. The mixture was vortexed and incubated for 15 min at 37°C. Following the incubation, the mixture was cooled in an ice bath and 400 µl of a color reagent (44 mM 3,5-dinitrosalicylic acid, 200 mM KOH, and 20 mM sodium potassium tartarate) was added. The mixture in glass tubes was covered and lowered into a boiling water bath for 25 min followed by cooling and the addition of 1.4 ml of distilled water. The mixture was then brought to room temperature and transferred to a plastic cuvette, and absorbance at 530 nm was measured with a spectrophotometer (Beckman DU-64).
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