这项工作是由国家卫生部授予DK083327 DK093491研究院（SZH）的支持。

1。准备胰腺腺泡细胞钙成像<ol><li>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。 </li><li>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）。 </li><li>准备胶原酶消化缓冲液中，加入1.1毫克/毫升（200单位/毫升）-4型胶原酶和1毫克/毫升大豆胰蛋白酶抑制剂的6毫升BSA孵育缓冲液（如上所述）。 </li><li>两部分硝酸和盐酸2小时的一部分浸渍酸洗22x22毫米的盖玻片。然后倒出使用去离子水并存储在70％的乙醇。 </li><li> CO 2窒息安乐死一个鼠标。东方动物在仰卧位，并准备在腹部表面由CLE70％乙醇aning。进行剖腹探查，腹腔暴露。胰腺解剖，在一个小的权衡船含胶原酶消化缓冲液6毫升，立即用清水冲洗。 </li><li>解剖任何大块的脂肪或可见血管，然后取出5毫升胶原酶消化缓冲液，并将其重新添加到15毫升锥形管。 </li><li>使用精细解剖剪刀切末胰腺小称量船胶原酶消化缓冲液含1毫升。剁碎，直到所得的溶液均匀地分散。 </li><li>碎的产品转移到一个125毫升的锥形塑料烧瓶中，然后加入剩余的5毫升胶原酶消化缓冲液的容器中。确保组织被完全淹没在缓冲区。 </li><li>将烧瓶放置在37℃水浴中，振摇30分钟90转。在这短短的停机时间，准备钙激活受体激动剂（ 如蛙皮素，卡巴）和冲洗22x22毫米酸洗盖DI水单。干燥，然后放置在平坦的表面上的顶部的实验室膜内衬。 </li><li> 30分钟的消化完成后，转让暂停回来到15毫升锥形管，使细胞定居。小心吸取胶原酶消化缓冲液和更换BSA孵育缓冲液6毫升。管用手大力摇晃，持续10秒，以使细胞分散成更小的簇。摇晃时立即删除任何大，漂浮物从悬而未决的媒体。 </li><li>允许剩余的细胞来解决，新鲜BSA孵育缓冲和交换媒体。 </li><li>重复步骤1.11两次，直至吊销只有细微到肉眼可见的小群组成。细胞应类似于图1A中所示的（顶行）。 </li><li>删除了BSA的孵育缓冲和更换与HEPES孵化缓冲。 </li><li>准备一个750微米，100X股票氟上午04时，在10％DMSO。 </li><li>将细胞在15毫升锥形管中，将细胞悬浮液和无BSA-HEPES孵育缓冲液。要做到这一点，添加合适的音量（1:100稀释）的原液细胞悬液。然后板500微升这种悬挂到每一个22x22毫米盖玻片。 </li><li>在室温下，在黑暗中保持盖玻片。以第一的Ca 2 +测量30分钟后加载。染料被自动起动时的灌注，发生30分钟后，染料吸附量，从培养基中除去。 </li></ol> 2。测量胰腺腺泡细胞钙<ol><li>用DI水冲洗每个灌注设置，并用适量的缓冲或激动剂。总理缓冲区或激动剂每个注射器，以确保适当的流量。夹注射器环站约1-2英尺以上的显微镜阶段。 </li><li>用细镊子仔细掌握其边缘盖玻片含细胞悬液。盖倾斜滑45°，让多余的缓冲地跑了出去。顶部的橡胶垫圈与盖玻片上的细胞的盖玻片放置，装配腔（ 图2A和2B）。 </li><li>灌注室固定在舞台上，并插入油管包含缓冲室进入第一入口（ 图2C）。将含有缓冲液的注射器上，并允许缓冲液灌注在整个表面上的盖玻片。一旦缓冲区已达到的腔室的另一端，插入了真空线到真空进样口。任何其他管（含激动剂，抑制剂等 ）插入到相应的位置沿室。 </li><li>可视化的细胞，使用一个200X或400X，1.4数值孔径的目标和氩激光激发萤光上午04时的染料，在波长为488 nm（ 图1A，下图）。的激光的功率设置应进行调整，使光电倍增管（PMT）是不饱和的发射光。此外，PMT的两端的电压，需要进行优化，以最大的光检测。 </li><li>大于515 nm的长通的发射信号被收集在帧速度为2-5秒/帧的可视化用于可视化的速度，全球钙波（ 图3和4）的缓慢振荡模式和0.2-0.3秒/帧。 </li><li>成像完成后，关闭注射器和删除所有的管子。拆卸室，并重复步骤2.2-2.4。 </li><li>荧光强度随着时间的推移和转移到图像J的数值（由美国国立卫生研究院免费提供的软件包，可以发现在<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> ）收集数据。 </li><li>查看实时蜂窝图像识别地区的利益（投资回报， 即心尖，基底核等 ） </li><li>采集荧光强度，这些投资回报率和represent荧光强度/基线荧光强度（ 即 F/F0）的。 </li><li>通过绘制F / F 0与生成描时间。除了描记，代表图像通常使用伪显示。 </li></ol> 3。胰腺腺泡细胞制备细胞损伤分析<ol><li>暖DMEM F-12媒体（不含酚红）至37°C。 </li><li>添加BSA（0.1％w / v的末期），和HCl（50μM最终）的DMEM F-12介质中。 </li><li> 50毫升的DMEM培养液分装成一个圆锥形管。 </li><li>准备一个缓冲区胶原酶IV型胶原酶0.5毫克/毫升（12单位/毫升），加入10毫升补充的DMEM F-12介质。 </li><li> CO 2窒息安乐死一个鼠标。东方动物在仰卧位，并准备用70％乙醇清洗腹部表面。进行剖腹探查，腹腔暴露。胰腺解剖，并立即用清水冲洗，一个小称量船Çontaining 6毫升胶原酶缓冲区中。 </li><li>剁碎组织使用精细解剖剪刀3-5分钟或直到出现所得溶液均匀地分散。 </li><li>碎组织转移到一个125毫升的锥形烧瓶中加入额外的的胶原酶缓冲区，然后将用5毫升到37℃水浴中振荡5分钟，以90 rpm。 </li><li>从摇床中删除，使细胞简要定居，并交换用5毫升的新鲜胶原酶缓冲。然后孵育一个额外的35分钟，在37℃水浴振荡在90转。 </li><li>大力悬浮消化，用移液管停牌，直至出现均匀无明显的细胞团块。然后轻轻移液管将细胞悬浮液通过一个预湿的尼龙网丝成锥形瓶中。 </li><li>大力吸取额外的3毫升新鲜的DMEM F-12介质（用胶原酶），通过网孔，，以迫使任何剩余的细胞通过。 </li><li>添加另一个6-10毫升的DMEM F-12媒体和LLOW细胞下沉2-3分钟。重复此步骤两次，最后一次洗涤后，去除上清。 </li><li>新鲜的DMEM F-12介质，悬浮细胞，和板500μl的细胞悬浮液添加到48孔组织培养板的各孔中。的细胞应类似于图1B中所描述的那些。 </li><li>允许坐板在37℃水浴90转5分钟，然后再添加激动剂。 </li><li>刺激细胞不同激动剂2-4小时。 </li><li>倾斜板成一角度，以使细胞沉淀，并仔细吸取介质（通常为100微升）的等分试样和闪光灯在液氮中冷冻。 </li><li>闪光冻结剩余的细胞悬液。闪光冻结是没有必要为LDH测量。作为一种替代的样品可以保存在冰中，如果他们将被测定的当天，或储存在-20℃下长期贮存。 </li></ol> 4。测量乳酸脱氢酶泄漏<ol><l&gt;测量乳酸脱氢酶（LDH）活性，主要使用Promega公司利用CytoTox 96非放射性细胞毒性检测试剂盒（见表特定的试剂和材料）中提供的试剂和说明。 </li><li>每瓶12毫升测定缓冲液（试剂盒中提供的），乳酸脱氢酶（LDH）衬底（亦提供）。 </li><li>解冻冷冻的细胞悬浮液和培养基样品在室温下在水浴中。 </li><li>板50微升在96 - 孔板的每一个媒体样本。 </li><li>在细胞悬浮液中，添加必要的10倍体积的裂解缓冲液，使最终浓度为1X。轻轻振荡后，在37℃60分钟。 </li><li>溶解的样品在250×g离心4分钟，沉淀细胞碎片。 </li><li>对于细胞裂解物，板加入50μl1:10稀释到96孔板的各孔中。 </li><li>重组后的基板向每孔中加入50μl，在室温下在黑暗中孵育板30分钟。 </li><li>停止反应，终止液（在试剂盒提供），向每孔加入50μl。 </li><li>测量在490nm处的吸光度。 </li><li>使用下列公式计算％LDH漏出。减去一个空白的OD值的读数从一个仅含有PBS中，衬底的下面的光的密度进行校正，并停止溶液。 </li></ol> LDH媒体=（OD492媒体）×（媒体稀释倍数）×（第一卷媒体）*由于它通常是不必要的媒体样本稀释，稀释因子将最经常equal1的。 总LDH =（（裂解液OD 490）×（裂解液稀释倍数）×（第一卷细胞悬液））+（（媒体外径490）×（稀释倍数）×（分装取样媒体第一卷）） <img alt="公式1" fo:src="/files/ftp_upload/50391/50391eq1highres.jpg" src="/files/ftp_upload/50391/50391eq1.jpg" /> 5。胰腺腺泡细胞感染腺病毒<ol><li>准备胰腺腺泡细胞在第3节。 </li><li>步骤3.12，新增10 7传染性单位（IFUs）的腺病毒的细胞悬液孵育30分钟，在37°C </li><li>均匀板2 ml的细胞悬液，在6孔板中。 </li><li>对于大多数表达结构，细胞应充分感染18小时内和一些荧光素酶的结构，只是在6小时内。 </li></ol>

<ol>
	<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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+isolated+pancreatic+exocrine+cells.">Structural and functional characterization of isolated pancreatic exocrine cells.</a> Proc. Natl. Acad. Sci. U.S.A. 69, 3028-3032 (1972).</li><li>Van Acker, G. J., 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=Tumor+progression+locus-2+is+a+critical+regulator+of+pancreatic+and+lung+inflammation+during+acute+pancreatitis.">Tumor progression locus-2 is a critical regulator of pancreatic and lung inflammation during acute pancreatitis.</a> J. Biol. Chem. 282, 22140-22149 (2007).</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=Kinetic+control+of+multiple+forms+of+Ca(2+)+spikes+by+inositol+trisphosphate+in+pancreatic+acinar+cells.">Kinetic control of multiple forms of Ca(2+) spikes by inositol trisphosphate in pancreatic acinar cells.</a> J. Cell Biol. 146, 405-413 (1999).</li><li>Leite, M. F., Burgstahler, A. D., Nathanson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++waves+require+sequential+activation+of+inositol+trisphosphate+receptors+and+ryanodine+receptors+in+pancreatic+acini.">Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini.</a> Gastroenterology. 122, 415-427 (2002).</li><li>Paredes, R. M., Etzler, J. C., Watts, L. T., Zheng, W., Lechleiter, 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=Chemical+calcium+indicators.">Chemical calcium indicators.</a> Methods. 46, 143-151 (2008).</li><li>Schild, D., Jung, A., Schultens, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+calcium+entry+through+calcium+channels+in+olfactory+receptor+neurones+using+a+laser+scanning+microscope+and+the+calcium+indicator+dyes+Fluo-3+and+Fura-Red.">Localization of calcium entry through calcium channels in olfactory receptor neurones using a laser scanning microscope and the calcium indicator dyes Fluo-3 and Fura-Red.</a> Cell Calcium. 15, 341-348 (1994).</li><li>Saluja, A. K., 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=Secretagogue-induced+digestive+enzyme+activation+and+cell+injury+in+rat+pancreatic+acini.">Secretagogue-induced digestive enzyme activation and cell injury in rat pancreatic acini.</a> Am. J. Physiol. 276, 835-842 (1999).</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=Ryanodine+receptors+contribute+to+bile+acid-induced+pathological+calcium+signaling+and+pancreatitis+in+mice.">Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. , (2012).</li><li>Gurda, G. T., Guo, L., Lee, S. H., Molkentin, J. D., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholecystokinin+activates+pancreatic+calcineurin-NFAT+signaling+in+vitro+and+in+vivo.">Cholecystokinin activates pancreatic calcineurin-NFAT signaling in vitro and in vivo.</a> Mol. Biol. Cell. 19, 198-206 (2008).</li></ol>

没有利益冲突的声明。

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。 <...

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。

<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

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。

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...

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

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。

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

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 次观看.  Children's Hospital of Pittsburgh of UPMC. 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。

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

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

科研

生物学

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

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.

评估胰腺腺泡细胞的分泌能力

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

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

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.

小鼠胰腺胰岛细胞亚群中的钙动力学成像

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