制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染
Summary
我们描述一个可重复的方法准备从小鼠小鼠胰腺腺泡细胞研究生理和病理相关刺激腺泡细胞钙信号和细胞损伤的目的。这些细胞中腺病毒感染的方法也被提供。
Abstract
胰腺外分泌胰腺细胞是主要的实质细胞，进入胰管和胰腺酶的分泌起着主要作用。这也是网站发起胰腺炎。在这里，我们介绍了如何腺泡细胞分离从整个胰腺组织及细胞内钙信号的测量。此外，我们描述的技术，与腺病毒构建体转染这些细胞，并随后测量乳酸脱氢酶，细胞损伤的标志物的泄漏，以及在体外诱导腺泡细胞损伤的条件。这些技术提供了强大的工具来表征腺泡细胞生理和病理。
Introduction
胞内钙离子的动态变化是必要的生理和病理腺泡细胞事件。这些不同的效果被认为是导致钙从不同的空间和时间模式的钙信号1。例如，从腺泡细胞分泌的酶和流体与钙尖峰其中分泌发生根尖极受限制的区域。与此相反，一个全球性的钙波强烈的非振荡钙信号与早期导致急性胰腺炎3,4的病态事件。这些措施包括腺泡内的蛋白酶激活酶的分泌减少，腺泡细胞损伤。我们的实验室使用孤立的胰腺腺泡研究这些早期病理事件，导致疾病在体内和体外 5-7。这里详细描述的方法为目的的测量CY隔离主要腺泡细胞tosolic钙水平和细胞的损伤。这些细胞中腺病毒感染的方法也被提供。
Protocol
1。准备胰腺腺泡细胞钙成像准备HEPES孵育缓冲液含有20mM的HEPES，95 mM氯化钠，氯化钾4.7毫米，0.6毫米MgCl 2，1.3毫米的 CaCl 2，10mM葡萄糖，2mM谷氨酰胺，1×最小的Eagle培养基非必需氨基酸。调整最终的pH值至7.4，用NaOH溶液。 准备一个BSA孵育缓冲液，HEPES培养缓冲液（如上所述），以25毫升，加入牛血清白蛋白（1％w / v的末期）。 准备胶原酶消化缓冲液中?…
Representative Results
腺泡细胞钙测量的生理刺激的反应的一个例子在图3中提供。腺泡细胞钙装有染料和荧光4灌注乙酰胆碱模拟氯化氨甲酰胆碱（CCh的1μM））8。细胞的反应中的钙波的形式，从而启动在顶端区域，并传播到在基底外侧区域3,9。 如图3B中所示的代表轨迹的呈现出的典型的峰值高原通常观察1μM的氯化氨甲酰胆碱的图案。相反，使用次最大剂量的胆囊收缩素类似物…
Discussion
细胞分离方法和随后的分析这里描绘了强有力的工具来研究生理和病理的胰腺外分泌功能。隔离分散胰腺腺泡细胞的方法最早是由阿姆斯特丹和贾米森在1972年11。这里介绍的方法已经适应从更近的隔离方法由范·阿克尔和同事12。虽然这些技术是高度可重复性，也容易学习的，有几个重要的功能，必须进行精确的每个方法。了解这些技术的细节将允许更容易故障排除和改进应用。 <…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家卫生部授予DK083327 DK093491研究院（SZH）的支持。
Materials
| 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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Cite This Article
Orabi, A. I., Muili, K. A., Wang, D., Jin, S., Perides, G., Husain, S. Z. Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection. J. Vis. Exp. (77), e50391, doi:10.3791/50391 (2013).