这项工作是由健康研究加拿大学院资助[MOP-1112666至CVB＆ME]支持。

伦理学声明:所有实验和程序与不列颠哥伦比亚省，温哥华，加拿大大学的实验室生物安全指导方针达成协议进行。 1. 35毫米玻璃底培养皿准备基于FRET的共聚焦显微镜<ol><li>使用以下步骤36-48小时前实验制备平板。 </li><li> -20℃存储检索选择胶状蛋白质混合物和0.25％胰蛋白酶-EDTA，并保存在移动冰浴中，直到使用。 注:包衣蛋白混合物应该只暴露于室温的时间，以防止该混合物从使用前固化短暂。直到需要胰蛋白酶溶液也应保持冷藏;可替代地，胰蛋白酶可为-20℃存储以下步骤1.5完成检索。 </li><li>准备1:25稀释蛋白混合物:DMEM（不添加血清，冷冻）。转运蛋白的混合物/ DMEM适当的体积正在准备每盘。 35毫米的玻璃底板，加入大约200微升完全覆盖在盘底内层玻璃圆。 </li><li>离开板坐安全柜内，并在室温至少30分钟。在这段时间内暖含10％NCS的到37℃在水浴中。 </li><li>接下来的步骤前夕温暖的胰蛋白酶在水浴至37℃。 </li><li>除去DMEM使用玻璃吸管和吸入当前正在培养瓶中。用温无菌PBS烧瓶洗楼，除去残存的DMEM残留物。 </li><li>逐出瓶底部的预先培养平滑肌细胞: 注:烧瓶成立天到这一步，以允许板制备前细胞以达到所需的汇合（约80％）。细胞最初悬浮在烧瓶的20ml DMEM中，用10％NCS和在37℃，5％CO 2的供给培养24小时，DMEM供应之后被刷新，并放回细胞培养箱中。 DMEM进行了改版前夕直到细胞达到汇合RY 48小时以下的初始步骤。 <ol><li>洗1毫升胰蛋白酶迅速瓶中，然后用吸拆下1个ml的胰蛋白酶。 </li><li>加在再过1 ml胰蛋白酶和离开较长时期（约30-60秒），嗖嗖烧瓶以确保酶使用该烧瓶的整个底部接触。 </li><li>观察有多少细胞已经在显微镜下分离直至满意与瓶分离。备用嗖嗖胰蛋白酶溶液和检查细胞脱离，直到满意与瓶分离的细胞比例。 注:将烧瓶可以返回到培养箱中约30-60秒，以加快在37℃下此过程。 </li><li>一旦细胞已被使用胰蛋白酶脱落，用10％NCS的添加新鲜的DMEM向烧瓶以停止胰蛋白酶反应（足够容积，使胰蛋白酶形成1/8在烧瓶总体积）。 注意:例如，使用250毫升的培养瓶中时，这会为7的10ml DMEM加到1 ml胰蛋白酶的导致在8ml细胞溶液。 </li></ol></li><li>转让所得从烧瓶到一个干净的（标记的）50ml锥形管中的细胞溶液。 </li><li>加入1毫升的新鲜DMEM加10％NCS每块板正在准备（足以媒体要持续24小时）。 </li><li>轻轻颠倒含有管细胞溶液多次分手可能已经形成的任何沉淀。 </li><li>吸管所需轻轻混合细胞溶液到各板的体积。 注:镀金此协议的建议平滑肌细胞数量是0.5-0.8×10 6？35 100mm培养菜肴。这些数字可根据细胞类型而有所不同，应通过每个实验室进行测试和修改。 </li><li>旋流每块板彻底顺时针/逆时针，以确保整个玻璃底细胞的平等分配。 </li></ol> 2.瞬时转染与腺病毒载体携带D1SR 钙指标<ol><li>在加入我的dium和细胞溶液到每个平板，离开板在37℃，5％的CO 2供给孵育至少1小时，以允许细胞正常附着到玻璃上的菜肴底部。在15分钟的间隔在显微镜下检查板直至细胞被清楚地附着。 </li><li>检索来自-80℃下贮存的腺病毒 - D1SR等分和保持在移动冰浴输送到生物安全柜。 </li><li>吸管所需剂量冷冻D1SR到各板的（100感染复数）。 注:对于每板所需的腺病毒溶液的体积计算取决于在原始原料溶液，这是作为在噬斑形成单位（PFU）的病毒滴度。根据我们的经验，我们建议的100感染复数（MOI），它被解释为对感染的培养板一次平滑肌细胞（SMC）所需的数量或病毒颗粒的多重性。 </li><li>孵育感染的细胞在37℃，5％的CO 2供给O / N。 </li><li>第二天，补充细胞用1毫升新鲜的DMEM加10％NCS。 </li><li>在5％CO 2 O / N在37℃孵育，在湿润的培养箱板上。 </li></ol> 3. SR鲁米的Ca 2+测量使用基于FRET的共焦成像<ol><li>继O / N孵化，请从平板培养基。 </li><li>用含1毫升温生理的HEPES-PSS缓冲液洗涤培养板（在mmol·L - 1）的NaCl 140，葡萄糖10，氯化钾5，HEPES 5， 氯化钙 1.5和MgCl 2的1（pH7.4）中三次。 </li><li> 1毫升新鲜温暖的HEPES-PSS缓冲录制开始前立即添加。 </li><li>执行使用的共聚焦显微镜的相关软件的FRET SE（敏排放）的应用程序（或类似功能），最初的影像。 <ol><li>一旦应用程序打开，点击进入"设置"选项卡以调整设置以达到理想的实验条件。 </li> &lt;LI&gt;手动更改频道设置，使细胞的序列很高兴在440纳米（供体和FRET通道）和513 nm（用来受体通道）。在488纳米（供体）和535纳米（FRET为和受体）收集的发射波长。 </li><li>设置的光的强度在15％的透射率，与细胞（记录时间为三个连续的CFP给体，FRET，和YFP受体通道）和曝光之间10秒的时间间隔的150毫秒励磁曝光时间。 </li></ol></li><li>在显微镜舞台上稳定板。 </li><li>使用"实时"按钮，检查图像分辨率。进一步调整设置，直至达到所需的分辨率。 </li><li>仍然在"设置"选项卡下，在使用"捕获图像"按钮，样品的图像。 </li><li>移动到"评估"选项卡中，选择适当的方法来计算正在做的实验FRET效率。方法3，使用比例计算[E A（I）= B / A]，被推荐用于涉及cameleons实验如D1SR指示器。 </li><li>切换到"图形"选项卡，以便能够观察在实验过程中被记录在图表形式的强度值。 </li><li>画一个ROI在图像查看器来观察这个区域的图形作为实验所得利息相应的平均强度。 注:研究者还可以选择绘制多个ROI，并有相应的强度被记录并在图表上同时显示。可替代地，调查人员可能概述了初始记录单个投资回报率，并继续由该软件的数据取向分析版本打开实验文件概述在稍后的时间多个ROI。 注:建立基于FRET的实验所需的步骤的详细内容可在FRET应用程序向导手册中找到。如果在显微镜没有配备FRET的向导，以下方案可用于设置的参数手动录音。 </li><li>开始记录，并允许数据收集至少3分钟，以确保信号的一个稳定的基础记录引入感兴趣的试剂之前。 </li><li>引进的 Ca 2+ -moving剂（ 如 2微米毒胡萝卜素; 100nm的内皮素-1），作为消耗的SR的Ca 2+，通过手动直接作为邻近吹打预定剂量进入板在一个点的兴趣存在的区域中的工具记录为可能的。继续记录为一个希望的时间后处理。 </li><li>捕获比例FRET序列图像（512×512像素），使用FRET SE应用程序的共聚焦显微镜倒置的63X油浸物镜。 </li><li>收集成像造成5-10平滑肌细胞中感兴趣区域（ROI）的每个区域集群数据，然后格式化并使用基于统计的分析软件。 <ol><li>要保存供以后使用的数据，在右键单击该记录的跟踪，并选择"导出"选项SAVE中的电子表格与研究者的选择驱动器上记录的平均信号强度。 </li><li>通过将每个数据点由在时间点0秒观察到的起始强度值正常化从每个单独的实验数据。 </li><li>通过手动复制并从原来的电子表格/秒粘贴相关数据行从电子表格到一个统计程序的项目文件导入规范化的数据。 </li><li>自动生成对应于粘贴到程序中的数据集的曲线图。 注:该程序的默认设置生成导入的数据线图。显示的图表类型可以通过点击下面的"更改"的主要工具栏中找到航向"选择不同类型的图形"按钮进行修改。 </li><li>代表一个较大基团的相同实验中的多个独立试验成单个数据片池数据以产生用于执行的特定测试的平均跟踪。 </li></ol></li></o升&gt; 4.准备细胞质的Ca 2+指标<ol><li>在1ml的黑色微量离心管二甲基亚砜（DMSO）的溶解0.0125克聚醚酸（PA）（可能需要暖管中流动水浴，以帮助溶解PA）。 </li><li>检索来自-20℃下贮存的胞质钙指示剂染料（的Fluo-4 AM）的一个管（50微克）。 </li><li>包裹染料的管在箔来保护它尽可能从与光接触。 </li><li>吸移管45微升的DMSO + PA溶液到含有染料的管构成。裹在室温在箔和存储DMSO + PA解决方案。 </li><li>吹打的解决方案上下多次调匀管的内容。可替代地，超声处理的染料管10分钟。 注:在整个DMSO + PA解决方案足以彻底分配染料无论哪种方法;它仅仅是研究者的偏好的问题。如果研究者选择超声处理解决方案，应的一隔离区/房间，其他人会受到噪声的影响来完成。研究者还应该使用重型消声器，以保护耳朵。 <ol><li>如果超声解决方案，将铝箔包裹管含发泡管座和地方的2/3（与卫生署2 O）超声器里面洗澡里面的染料。 </li><li>在隔离区/室和动力机在地方超声波仪浴（确保保护帽到位启动超声处理之前）。留有余地，离开机器关了之，检索管（保持在铝箔包裹避光，只要可能的话）之前，运行10分钟。 </li></ol></li><li>分装现在充分混合溶液到暗离心管（每管5微升，应该能够使10管总数）。 </li><li>包装在铝箔等分各管。标签管并存储在干燥剂在-20°C条件下直至使用。 </li></ol> 5.细胞质的Ca 2+测量<ol><li>按照1.1-1.12步骤准备大鼠主动脉平滑肌细胞培养板。 </li><li>从在48小时后培养板除去培养基。 </li><li>除去-20℃存储胞质的 Ca 2+指示剂（5微升）的预先制备的等分试样，并在移动冰浴保持直至使用。 </li><li>用含1毫升温生理的HEPES-PSS缓冲液洗涤培养板（在mmol·L - 1）的NaCl 140，葡萄糖10，氯化钾5，HEPES 5， 氯化钙 1.5和MgCl 2的1（pH7.4）中三次。 </li><li>混合指示剂用1毫升温HEPES-PSS的，它加载到培养皿。 </li><li>孵育平滑肌细胞与在RT指示器1小时（24℃）。 </li><li>从培养板去除指示器，并用1毫升温生理的HEPES-PSS缓冲三次洗涤。 </li><li> 1毫升新鲜温暖的HEPES-PSS缓冲录制开始前立即添加。 </li><li>捕捉序列图像（512×512像素），具有63XØ共聚焦显微镜倒置的IL-浸泡的目的。 <ol><li>手动改变设置，使得细胞被激发在488nm，和发射波长在700赫兹的扫描速度555nm处收集。 </li><li>设置的光的强度在15％的透射率，与细胞的150毫秒励磁曝光时间（记录过程中的细胞暴露于光以指定的时间间隔的时间量）和曝光之间5秒的间隔。 </li></ol></li><li>稳定在显微镜舞台板，并开始录制。 </li><li>记录至少3分钟，以确保信号的一个稳定的基础记录引入感兴趣的试剂之前。 </li><li>引进的 Ca 2+ -moving剂（ 如 2μM毒胡萝卜素），作为消耗的SR 的 Ca 2+（如在步骤3.16中所述），并继续录制时间后处理的所需量的工具。 </li><li>使用特定显微镜的软件通过平均荧光SI记录信号强度gnals（F / F 0）从5-10平滑肌细胞中感兴趣区域（ROI）的每个区域的群集。收集，格式和分析使用数据分析软件从成像结果数据。请参阅使用本软件的步骤3.18.1-3.18.5用于数据收集的描述和分析。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

这个协议描述平滑肌的转染与D1SR腺病毒到SR的内腔中研究的 Ca 2+的浓度。此方法允许的 Ca 2+的此细胞器中的直接测量，以及它的运动进/出SR的不同钙移动剂的应用的结果。该方法对朝更改SR和胞质钙离子水平如何影响整体的健康细胞，以及这些变化是相互关联的一个全面的了解调查工作的许多优点。细胞内Ca 2+的移动的传统观测涉及改变仅细胞质<su...

钙离子是人体的每一个细胞中大量存在一个非常重要的离子。它有许多不同的细胞功能，包括生长，增殖，迁移，凋亡和1-4显著的作用。这是公认的Ca 2+可以通过直接和间接的行为影响这些过程，因此，改变该离子的正常细胞内浓度可以很容易地得到用于受影响的细胞中的负面结果。的SR被认为是细胞5内的最大细胞内Ca 2+存储。同时在SR和胞质钙离子稳态水平，通过不断变化进入和退出这个细胞器的钙 -transporting渠道的正常维护。由于任何形式的伤害或疾病在细胞施加的压力条件通常与SR钙离子的去到对长期影响他显著变化相关细胞ALTH。在最严重的情况下，一个强调细胞无法收回，并保持稳定状态SR钙离子水平甚至可能死亡的细胞凋亡6-8高潮。 目前的研究到细胞钙动力学的事实，一些研究试验细胞器的 Ca 2+存储内容直接9-11限制。最常见的做法，而不是涉及SR 钙含量12-14胞质钙离子水平的变化间接测量的测量。在这些实验中，钙离子通常诱导从SR通过使用引起细胞器的耗尽药剂（ 如毒胡萝卜素）的释放。结论然后就基于在细胞质的 Ca 2+浓度的波动变化到SR 的 Ca 2+绘制。尽管剩余研究者以一种迂回的马得出这样的结论的能力nner，SR 钙这种测量方法显然是来收集这些信息，提供关于收集的数据的解释很多局限性一种间接的方式。为了绕过此明确的限制，有必要测量的 Ca 2+的SR管腔网络内直接发现的量。 至关重要的是能够直接记录腔内的SR 的 Ca 2+水平的最终结果是细胞培养工具和所用的钙离子指 ​​示剂。在当前的手稿中引用的数据，必须指出，使用血管平滑肌细胞从冷冻的细胞系来是很重要的。细胞在Dulbecco改进的Eagle培养基（DMEM）+ 10％新生小牛血清（NCS）以上的概述实验的通道22-26，在一个恒定的37℃，5％CO 2的供应孵育培养。使用当前的方法，例如，细胞已经非常成功地生长在模拟胞外蛋白质混合物许多不同类型的组织15的环境。重要的是沿着SR Ca的静脉成功2+研究是用于蛋白质混合物的类型;在这种情况下，一个低生长因子各种必要避免从影响常规的Ca 2+信号和运动不断测试的细胞内发生的该工具的组件。以下的试验细胞的成功的增长，所述钙离子指 ​​示剂也必须有效地引入到这些培养细胞。这已经通过使用携带SR-驻留的 Ca 2+指示剂D1SR的腺病毒载体成为可能。实现了高转染效率，细胞必须用病毒载体孵育成像前至少36-48小时。该制剂提供了测量的Ca以高精度和再现性的SR腔内2+瞬变的可靠工具。 在这个协议中使用D1SR指标是D1ER钙的改良变种2+我最初由钱永健博士的实验室在加州大学圣地亚哥分校，美国9,16创建ndicator。原始D1ER属于第二代的遗传编码的 Ca 2+指标称为cameleons，超过一个更广泛的范围（0.5-160微米）显示的 Ca 2+敏感性与先前的 Ca 2+指标9,16。该新变种D1SR，韦恩Chen博士（加拿大阿尔伯塔大学）是一种恩赐，但是，携带突变集钙蛋白序列（而不是钙网蛋白序列原D1ER）。突变体肌集钙蛋白具有结合降低的 Ca 2+，消除在对SR管腔11内结合的 Ca 2+内源性肌集钙蛋白竞争的问题。该D1SR指标进行截断的增强型青色荧光蛋白（CFP）和黄色荧光蛋白（YFP），其结合含改性钙调素（CAM）和一个M13连接蛋白（T他26个残基结合CAM）序列的肌球蛋白轻链激酶的肽。凸轮M13序列已被修饰以防止M13的结合内源性钙调蛋白。同时，为了确保SR滞留，一个集钙蛋白序列已经添加CFP 11的5&#39;端。当结合的 Ca 2+，凸轮-M13域经过导致的增加的侧翼CFP和YFP，其被记录为一个增加的FRET信号强度之间的能量传递的构象变化。另一方面，当SR管腔的 Ca 2+的浓度下降，凸轮-M13域经过反向构象变化，从而导致在侧翼CFP和YFP，并在FRET信号强度的显著降之间的能量传递减少。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (high glucose, pyruvate)</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix Growth Factor Reduced, 5mL</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>Newborn Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>35mm glass-bottomed plates</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL 0.2 &#956;M vented blue plug seal cap flask</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solution</td>
			<td>Life technologies</td>
			<td>25200056</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>D1SR</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Gift</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8779</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4-AM</td>
			<td>Life technologies</td>
			<td>F-23917</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>1.5 mL Black Microcentrifuge Tubes</td>
			<td>Argos Technologies</td>
			<td>T7100BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite 2.6.3 (with FRET SE Wizard application)</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.0</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of calcium fluctuations within the sarcoplasmic reticulum of cultured smooth muscle cells using fret-based confocal imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

本部分提供了可以使用上述方法来捕获的SR管腔Ca 2+浓度的数据来获得的结果的例子，相对于间接测量的变化，通过观察在细胞质的Ca波动细胞器的 Ca 2+商店浓度通常采用的方式2+ 。 图1A示出的与YFP通道（514 nm激发/ 535nm处发射）D1SR腺病毒转染的SMC培养的感兴趣区域（ROI）的区域的?...

Watch this Scientific Journal Video about Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging at JoVE.com

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

measurement-calcium-fluctuations-within-sarcoplasmic-reticulum

Research

JoVE Journal

Biology

7.6K 次观看.  University of British Columbia. Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

视频: Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

科研

生物学

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

人脐动脉平滑肌细胞的分离（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

肺动脉平滑肌细胞从新生小鼠隔离

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...