我们感谢这个手稿的阅读和编辑医生诺亚魏斯勒德。这项工作得到了香港研究资助UMDNJ基金会62-09透明带，美国心脏协会SDG2630086到新征，0535555N到MB，国立卫生研究院RC2AR058962-01到MB。

1。细胞内Ca 2 +测量单个细胞<ol><li> KYSE-150，一个人食管鳞状细胞癌（ECSS）细胞株培养在5％CO 2的气氛，在37°C的混合的RPMI 1640/Ham的F-12培养基（1:1），含5％胎牛血清。 </li><li> KYSE-150细胞转染质粒或者含有shRNA的专门针对Orai1或炒序列。质粒基因编码作为记者，这是由一个独立的启动子驱动的红色荧光蛋白（RFP），还包括。 </li><li>细胞生长在玻璃底菜（第1.5玻璃盖，MatTek，马）48小时。 </li><li>取出介质和冲洗细胞，用平衡盐溶液（BSS）（140毫米氯化钠，氯化钾2.8毫米，2毫米氯化钙2，MgCl 2的 2毫米，10毫米肝素钠，pH值7.2）。 </li><li>加入1毫升的BSS解决方案，包含2微米的Fura-2乙酰酯（分子探针）入菜，并用铝箔纸包裹，整道菜蛋白酶CT光。 </li><li>在37°C孵育40分钟的细胞，然后离开他们在室温下为一个额外的15分钟内让酯水解完成。与BSS洗涤细胞两次。 </li><li>装入菜尼康TE200倒置显微镜，它是连接公共交通交汇处分光光度计激发波长，在350和390 nm和510 nm处的发射阶段。要使用的确切的激发波长可以略有不同取决于各个系统的光学。确定最佳比例动态两个波长激发频谱扫描的Fura-2盐中含有的Ca 2 +从0到39.8μM（或更高浓度的Fura-2荧光饱和）的解决方案。 </li><li>成立了4个不同的解决方案在BSS重力灌注系统的Ca 2 +（2毫米），EGTA（0.5毫米），EGTA-TG（毒胡萝卜素，5μM）的Ca 2 +-2-建业（SOCE抑制剂"，75 μM）计算机控制的自动化perfusi系统上也可以使用。 </li><li>选择细胞表达RFP的可视化模式。 </li><li>定位在10倍放大倍率的灌注系统启用提示，直到针尖以上地区的利益是在45度角。 </li><li>同时记录的F 为350nm和F 390nm荧光信号。比（F 为350nm / F 390nm）显示，在第三个窗口。更改灌注以下顺序的解决方案的Ca 2 +; EGTA的Ca 2 + EGTA-TG的Ca 2 +的Ca 2 +-2-建业。 </li></ol>上述协议可以修改为测量细胞内Ca 2 +在细胞悬液系统。 <ol start="12"><li>文化KYSE-150细胞在与上述相同的培养条件下的T25瓶。 </li><li>当细胞达到90％汇合，除去介质和冲洗细胞与BSS解决方案。 </li><li>添加2.5毫升含2微米的Fura-2 AM和孵化的BSS解决方案细胞40分钟，在37°C间跟着脱酯化。 </li><li>删除的BSS，添加2.5毫升胰蛋白酶在37°C，直到细胞分离到烧瓶，并培育。再加入2.5毫升培养基停止胰蛋白酶和收获的细胞，在800转离心5分钟。 </li><li>离心洗更多的时间与培养基中的细胞重新悬浮颗粒细胞的BSS解决方案（不加2毫米氯化钙2，含有μm范围内的Ca 2 +）和计数细胞使用hematometer数。 </li><li>加入到石英比色皿（10毫米，Starna细胞）〜10 6细胞和填充量高达2毫升与BSS。 </li><li>放入分光光度计试管（与磁棒搅拌）。 </li><li>同时记录与发射波长在510 nm处的F 340nm的和F 380nm的荧光信号。正在运行的协议是:BSS的，除了0.5μLEGTA（0.25M股票），另外1μL毒胡萝卜素（甘油三酯（TG），10毫米股票），另外4μL的Ca 2 +（1米股票）。 </li></ol> 2。培养细胞中的锰淬火法<ol><li>执行的Fura-2激发波长扫描解决方案，包含不同的Ca 2 +浓度范围从0到39.8微米，以确定钙离子浓度等色点的独立波长。通常情况下，等色点或360纳米左右。 </li><li> KYSE-150细胞培养在玻璃底部的菜肴和加载的Fura-2 AM。 </li><li>成立灌注系统:与5个不同的BSS解决方案的Ca 2 +（2毫米），EGTA /易性癖（0.5毫米和5μm，分别），锰2 +（0.5毫米，不加EGTA或钙离子含有游离的Ca 2 +在μm范围内），锰2 + / 2-APB（75μM），EGTA /剂Triton X-100（0.1％）。 </li><li>装载显微镜阶段的菜，然后选择"激励比"模式，激发波长为360 nm和390 nm，发射，在510纳米。 </li><li>同时记录的F比390nm与360nm处和F（F 360nm处 / F 390nm）在第三个窗口显示的荧光信号。正在运行的协议是:1分钟的Ca 2 +稳定的荧光， 锰+ 30秒，10分钟，锰EGTA /易性癖2 + 2分钟，EGTA海卫X-100的（0.1％），持续1分钟获得的背景读数。可用于锰2 + / 2，建业解决方案，而不是对Mn 2 +检查取消的荧光猝灭，这表明了Mn 2 +入门SOCE途径是通过。 </li></ol> 3。在肌肉细胞中的锰淬火法<ol><li> C2C12细胞，小鼠肌细胞线，是在37°C的DMEM含10％胎牛血清，10％马血清的培养基培养玻璃底菜在5％CO 2气氛。 </li><li>细胞达到汇合后，改变培养基分化培养基（除去胎牛血清和减少é马血清2.5％）。后4天，C2C12细胞分化成肌管。 </li><li>加载C2C12肌管与5μM终浓度的Fura-2上午在1.4-1.6。 </li><li>添加终浓度为20μM的N-苄基-P-甲苯磺酰胺（BTS），来抑制肌肉收缩和运动造成的工件，并允许在开始实验前15分钟。 </li><li>交汇处的设备连接到显微镜的舞台上安装盘和灌注系统装入以下BSS解决方案:2的Ca 2 +（2毫米），0的Ca 2 +，锰2 +（0.5毫米）;锰2 + / TG（0.5毫米，20μM，分别）。 </li><li>成立如上所述灌注系统。 </li><li>在第三个窗口中显示的比率（F 360nm处 / F 390nm），同时记录的F 360nm处和F 390nm荧光信号。正在运行的协议是:2的Ca 2 + 1分，0的Ca 2 + 1分钟FLUSH距离的Ca 2 +，锰2 +为0.5分钟，锰2 + / 10分钟，和Mn 2 TG + / EGTA的Triton X-100（0.1％）。 </li></ol> 4。空间和时间上解决皮肤肌纤维SOCE <ol><li>准备以下的解决方案。 等渗缓冲台氏:140毫米氯化钠，氯化钾5毫米，10毫米的肝素钠，MgCl 2的 2毫米，2.5毫米氯化钙2，pH值7.2，290 mosm 培养基:2％和1％的马血清青霉素，链霉素的DMEM培养液 解决方案＃1:109.6毫米的K-谷氨酸， 洗净 2毫米EGTA氢氧化钾，6.7毫米MgCl 2的三磷酸腺苷，2毫米，6毫米的磷酸肌酸（CP）20毫米列印，N-二（2 -羟乙基）-2 -氨基乙酸（BES），氢氧化钾，pH值7.0 清洗解决方案＃2:140毫米的K-谷氨酸EGTA-KOH毫米，6.5毫米MgCl 2的 ，2毫米的三磷酸腺苷，CP的6毫米，20毫米的BES，氢氧化钾，pH值7.0，5 剥皮的解决方案:140毫米的K-谷氨酸，MgCl 2的 6.5毫米，2毫米的三磷酸腺苷，CP的6毫米，20毫米的BES，氢氧化钾，pH值7.0 TT加载解决方案:90.6毫米，18毫米谷氨酸钠， 氯化钙 0.55毫米，2毫米EGTA-KOH，MgCl 2的 6.7毫米，5.4毫米ATP，15毫米的CP，0.0025毫克/毫升肌酸激酶（CK），20毫米的K-谷氨酸百世氢氧化钾，5微米羰基氰化物P-trifluoromethoxyphenylhydrazone（FCCP），PCA，pH值7.0 7.0 SR装载解决方案:107.8毫米，0.98毫米氯化钙2，EGTA-KOH 2毫米，6.6毫米MgCl 2的 ATP的5.4毫米，15毫米的CP，0.0025毫克/毫升对照，20毫米的BES-氢氧化钾，5微米FCCP的K-谷氨酸， pH值7.0，6.6前列腺癌 简消耗的解决方案:K -谷氨酸，100毫米40毫米10毫米谷氨酸钠，EGTA-KOH，10毫米1,2 -双（邻氨基苯氧基）乙烷-N，N，N&#39;，N&#39;-四乙酸（BAPTA ），0.35毫米MgCl 2的 ATP的0.5毫米，1毫米，20毫米的BES-氢氧化钾的CP，5微米FCCP，pH值7.0。 25毫米caffeine/20μM的毒胡萝卜素（TG），实验前加入。 </li><li>解剖的趾长伸肌（EDL）的肌肉，先放下老鼠，并安排在腿侧卧位，然后从脚踝到膝盖皮肤，切浅表肌肉层组织揭露了EDL，并断绝上下肌腱释放完整的EDL肌;重要的是保持尽可能长的肌腱。 EDL是转移到台氏液，含0的Ca 2 +和EGTA 0.1毫米，以防止任何收缩。 </li><li>立体显微镜下，用两个镊子保持较低的插入肌腱，并分成两束的EDL肌。重复的过程，以获得4年，然后8束。肌腱保持在该进程的结束，8束，这是关键。如果他们失去了一些捆绑，抛弃他们。 </li><li>立体显微镜下，每个EDL捆绑带是抱怨道，两筋和仔细拉长，直到3〜4分离的单个肌纤维都留下完整与肌腱两侧。 </li><li>将1滴的Ca 2 +在中间的玻璃底菜免费台氏缓冲直线上的菜，放下了EDL带，尽可能的快速清除解决方案，然后都用防水胶带，确保磁带的筋向下的磁带是严密的。先洗纤维0的Ca 2 +溶液中，然后用2.5毫米的Ca 2 +溶液的2倍。如果纤维超合同和损害注意到，丢弃的纤维。 </li><li>如果需要，可以培养的光纤长达96小时，使遗传操作。完整的培养基洗纤维的3倍，并加入2毫升入菜的介质，在5％CO 2和37°C间孵化器的地方。每次实验前，检查肌肉纤维和丢弃那些没有明确的条纹或污染的迹象，或如细胞膜膜挛缩损伤的迹象。良好的纤维回应，氯化钾，电刺激，刺激和咖啡因。 </li><li>单肌纤维的洗涤与洗涤液＃1的3倍和沐浴Rhod-5N 500微米和0.5毫米的Ca 2 + 5分钟内像剥皮的解决方案。 </li> &lt;李&gt;使用钨针连接到针座，机械立体显微镜下皮肤的纤维，使横小管（TT），自动重新密封，并捕获与Ca 2 Rhod-5N共轭+在TT，两次洗皮肤纤维洗涤液＃2。在蒙皮过程中删除单元格的宽度小于25％时是最佳的机械剥皮过程。 </li><li>加载SR，皮肤纤维孵育20μM的FLUO-5N 1小时，在室温下，广泛使用一个额外的30分钟洗涤液＃2，孵育清洗，让皮肤含有的解决方案，完整的去酯化染料。 </li><li> 30分钟后，皮肤纤维60X客观的共聚焦显微镜下可视化。获得相应的染料（激发波长为543 nm和发射时间比570纳米Rhod-5N;激发在488 nm和排放量在530-560纳米荧光5N）波长的荧光图像。 regi每个染料装领域选择感兴趣的组件（投资回报）​​。 </li><li>实验的协议如下:TT -负荷为120秒，装载的SR-120，SR消耗400-750以消耗的SR的Ca 2 +存储的解决方案解决方案解决方案。在此过程中，Rhod-5N强度的变化（电汇的Ca 2 +）和5N荧光强度（简的Ca 2 +）的记录，创造一个相对荧光的变化，在TT和SR的时间当然决心。荧光强度的平均值，从多种纤维作进一步分析。在TT /简式结束时的最大负荷强度正常化至100％。 </li></ol> 5。代表结果我们审查KYSE-150细胞的SOCE活动，利用细胞内Ca 2 +测量（ 图2）。使用利福平作为记者，我们可以选择orai1含有特定的shRNA质粒转染对单个细胞的基因编码SOCEÇhannel。与转染质粒与含有争夺的shRNA（ 黑色线 ），敲Orai1减少的SOCE活动（ 红色曲线 ）的蛋白质结果。 SOCE KYSE-150细胞中也证实了Mn 2 +淬火法（ 图3）。激发波长为360 nm报告等色点的Fura-2，荧光是独立的Ca 2 +浓度。后甘油三酯完全耗尽的ER的Ca 2 +店，灌注的Mn 2 +导致在一个显着的荧光减少。整体SOCE活动可以衡量的Fura-2荧光强度显示出更积极的SOCE一个陡峭的斜坡下降速率，而浅的斜坡意义不太积极的SOCE。 2建业细胞荧光下降的斜率要浅得多，这表明，这种化合物被阻断SOCE活动。锰2 +淬火率进行了测定，从最初的10秒，淬火仍然没有饱和的线性范围的记录。 类似的锰2 +淬火法进行肌肉（ 图4）。在这种情况下，锰2 +与TG。而甘油三酯消耗的SR的Ca 2 +商店，荧光猝灭坡逐渐改变，直至达到其最大速率。 S形曲线，表明这些实验条件下的分级SOCE激活。 健康完好的文化单肌纤维表现出明确和统一的条纹，没有污染的迹象，没有收缩损伤的迹象。这些纤维能够响应电刺激或去极化的解决方案合同。在共聚焦成像，Rhod-5N染料剥皮纤维在TT车厢诱捕表明哺乳动物的特征双峰模式（visu就是靠在红色通道）。在加载SR FLUO-5N上午，穿插典型的SR模式是可见的（绿色通道）。 TT /简装载解决方案的皮肤纤维灌注后，Rhod-5N和Fluo-5N荧光强度增加;灌注SR枯竭的解决方案后，在SR车厢和TT的车厢荧光水平开始下降，表明紧耦合之间的SR的Ca 2 +商店枯竭和SOCE激活，分别为（ 图5）。 <img src="/files/ftp_upload/3415/3415fig1.jpg" alt="图1" /> 图1。激活存储操作的Ca 2 +进入（SOCE）。Orai1，孔隙通道形成单位，位于质膜（PM）和STIM1的， 一种 Ca2 +传感器，位于质或肌质网（ER / SR）的膜。时，ER / SR的Ca 2 +门店减少，由于封锁，ER / SR的Ca 2 +泵（电监会A）或通过IP 3受体或ryanodine受体的Ca 2 +释放，STIM1的被激活。激活STIM1的分子形成的补丁和诱导的聚集Orai1，从而进一步导致SOCE激活。 <img src="/files/ftp_upload/3415/3415fig2.jpg" alt="图2" /> 图2。测量KYSE-150细胞内Ca 2 +在细胞外液的外汇。0的Ca 2 +（0.5毫米EGTA）到2毫米的Ca 2 +并没有引起任何细胞内钙离子的变化。 5μM的甘油三酯在0的Ca 2 +沐浴液引起的被动的ER的Ca 2 +释放。耗尽后雌激素受体 ​​的Ca 2 +商店（10分钟），持续内Ca 2 +海拔SOCE，可以通过阻断SOCE抑制剂通过激活胞外Ca 2 +（2毫米）此外，如SKF-96365和2 -建业（数据未显示）。含有专门针对的shRNA质粒转染细胞orai1（红色）显示SOCE争夺序列（黑色）转染细胞相比，显着降低。 <img src="/files/ftp_upload/3415/3415fig3.jpg" alt="图3" /> 图3。锰2 +的SOCE淬火法在KYSE-150细胞的Ca 2 +独立的Fura-2（360 nm）的激发波长测定锰2 +（0.5毫米）的Fura-2荧光猝灭。细胞经5μM10分钟TG完全耗尽的ER的Ca 2 +商店。 +加法（虚线，在第10秒）后， 锰的Fura-2荧光衰减斜率代表，这是表示单位时间内的荧光（初始值的100％）减少百分之SOCE激活。最大淬火的荧光信号是建立在实验结束后，裂解细胞，0.1％的Triton X-100（0％）。 2-APB（75μM）处理的细胞表现出了浅斜坡，建议SOCE这KYSE-150细胞中的化合物被封锁。 <img src="/files/ftp_upload/3415/3415fig4.jpg" alt="图4" /> 图4。在肌肉细胞中发现锰2 +淬火法梯度SOCE激活。SOCE激活可以注册，而甘油三酯的消耗SR的Ca 2 +商店。同时应用的Mn 2 +（0.5毫米）和TG（20μM）诱导细胞内的Fura-2荧光（青色虚线显示最快的淬火点），这是明显的，从最初的锰2 +淬火梯度和S形下降率（蓝色虚线）。锰2 +淬火坡度为2-APB（75μM）处理的基底细胞水平几乎保持相同，这表明，SOCE抑制。 <img src="/files/ftp_upload/3415/3415fig5.jpg" alt="图5" /> 图5。空间和时间上解决皮肤的肌纤维SOCE。 （一）RHOD-5N盐加载到TT和FLUO-5N AM被加载到的SR（B）申请的Ca 2 +消耗的解决方案后，在TT和SR车厢荧光下降。 FLUO-5N荧光的损失迅速释放的SR的Ca 2 +含量和Rhod-5N荧光损失建议SOCE激活。

<ol>
	<li>Parekh, A. B., Putney, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+calcium+channels.">Store-operated calcium channels.</a> Physiol. Rev. 85, 757-810 (2005).</li><li>Ma, J., Pan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+activation+of+store-operated+calcium+channel.">Retrograde activation of store-operated calcium channel.</a> Cell Calcium. 33, 375-384 (2003).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORAI1+and+STIM1+deficiency+in+human+and+mice:+roles+of+store-operated+Ca2++entry+in+the+immune+system+and+beyond.">ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca2+ entry in the immune system and beyond.</a> Immunol. Rev. 231, 189-209 (2009).</li><li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat. Rev. Drug Discov. 9, 399-410 (2010).</li><li>Pan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunction+of+store-operated+calcium+channel+in+muscle+cells+lacking+mg29.">Dysfunction of store-operated calcium channel in muscle cells lacking mg29.</a> Nat. Cell Biol. 4, 379-383 (2002).</li><li>Shin, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retrograde+signal+from+calsequestrin+for+the+regulation+of+store-operated+Ca2++entry+in+skeletal+muscle.">A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle.</a> J. Biol. Chem. 278, 3286-3292 (2003).</li><li>Ma, J., Pan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctional+membrane+structure+and+store+operated+calcium+entry+in+muscle+cells.">Junctional membrane structure and store operated calcium entry in muscle cells.</a> Front Biosci. 8, d242-d255 (2003).</li><li>Pan, Z., Damron, D., Nieminen, A. L., Bhat, M. B., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+intracellular+Ca2++by+caffeine+and+ryanodine+induces+apoptosis+of+chinese+hamster+ovary+cells+transfected+with+ryanodine+receptor.">Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor.</a> J. Biol. Chem. 275, 19978-19984 (2000).</li><li>Hirata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+store-operated+Ca2++entry+and+altered+Ca2++release+from+sarcoplasmic+reticulum+through+silencing+of+junctophilin+genes.">Uncoupling store-operated Ca2+ entry and altered Ca2+ release from sarcoplasmic reticulum through silencing of junctophilin genes.</a> Biophys. J. 90, 4418-4427 (2006).</li><li>Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+resistance+to+fatigue+and+altered+calcium+handling+properties+of+sarcalumenin+knockout+mice.">Enhanced resistance to fatigue and altered calcium handling properties of sarcalumenin knockout mice.</a> Physiol. Genomics. 23, 72-78 (2005).</li><li>Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azumolene+inhibits+a+component+of+store-operated+calcium+entry+coupled+to+the+skeletal+muscle+ryanodine+receptor.">Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor.</a> J. Biol. Chem. 281, 33477-33486 (2006).</li><li>Launikonis, B. S., Barnes, M., Stephenson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+coupling+between+skeletal+muscle+store-operated+Ca2++entry+and+the+inositol+trisphosphate+receptor.">Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 2941-2944 (2003).</li><li>Shuttleworth, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+relationships+between+Ca2++store+mobilization+and+Ca2++entry+in+an+exocrine+cell.">Temporal relationships between Ca2+ store mobilization and Ca2+ entry in an exocrine cell.</a> Cell. Calcium. 15, 457-466 (1994).</li></ol>

没有利益冲突的声明。

虽然的Ca 2 +结合的Ca 2 +免费的Fura-2激发波长为340 nm和380 nm处，分别的最佳比例的Fura-2的动态范围为Ca 2 +浓度测量，可能会发生在其他波长在一个特定的显微镜系统。波长的这种变化通常是由于除了与各种光学元件的光学路径的变化。在这项研究中，FURA-2激发波长为350 nm和390 nm的决定FURA-2荧光光谱的分析。 细胞内Ca 2 +水平的细胞质结果从几个来源的平衡，包括细胞内Ca 2 +释放，细胞外Ca 2 +进入，以及在ER和细胞膜钙离子排斥机制。为了隔离从单向SOC介导的Ca 2 +涌入细胞内Ca 2 +释放和Ca + EXTR延髓的Mn 2 +淬火法都可以使用。锰2 +被称为是能够渗透到细胞通过SOCE，但防渗膜表面挤压或吸收钙的ER +泵。因此，荧光猝灭代表进入细胞的单向锰2 +流量测量估计SOCE激活程度。锰2 +淬火法等色点波长在这样的Fura-2荧光强度是独立的Ca 2 +浓度。为每个系统等色点应确定由光谱扫描。另外， 锰+淬火可以在任何两个波长，在这样一种方式，最终的价值是独立的Ca 2 +浓度13记录调整后的荧光。 为了获得的SOCE骨骼肌肉的活动空间和时间的信息，我们采用双染的方法，它允许同时measuremeNT SOCE活性和SR的Ca 2 +在皮肤成年哺乳动物的EDL肌纤维含量。 SR钙2的变化之间的关系+活动内容和SOCE可以绘制指示阈值和灵敏度SOCE激活枯竭的SR的Ca 2 +存储的。 TT加载的解决方案进行了优化，另外加载的T管与Ca 2 +和吸T型管和SR的装载解决方案旨在促进最大的SR的Ca 2 +负荷，另外加载的T-管〜500微米的Ca 2 +。 FCCP在TT /简加载的解决方案和SR消耗的解决方案，以消除从线粒体的影响。

特定的试剂和设备表: <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> RPMI 1640培养</td><td>敏达</td><td> 10-040-CV </td><td></td></tr><tr><td>火腿的的F-12 </td><td>敏达</td><td> 10-080-CV </td><td></td></tr><tr><td> DMEM培养液</td><td>敏达</td><td> 10-013-CV </td><td></td></tr><tr><td>玻璃底菜</td><td> MatTek </td><td> P35G-1.5-14-C的</td><td></td></tr><tr><td> FURA-2 </td><td> Invitrogen公司（分子探针） </td><td> F1221 </td><td> -20℃，密封紧密，避光，溶解到二甲基亚砜</td></tr><tr><td>荧光5N </td><td> Invitrogen公司（分子探针） </td><td> F14204 </td><td> -20℃，密封严密，从l保护IGHT </td></tr><tr><td> rhod-5N </td><td> Invitrogen公司（分子探针） </td><td> R14207 </td><td> -20℃，密封紧，轻保护</td></tr><tr><td>石英比色皿</td><td> starna细胞</td><td> 3 Q-10 </td><td></td></tr><tr><td>毒胡萝卜素</td><td> tocris </td><td> 1138 </td><td></td></tr><tr><td> 2 Aminoethoxydiphenylborane（2-APB） </td><td> tocris </td><td> 1224 </td><td></td></tr><tr><td> N-苄基-P-甲苯磺酰胺（基站） </td><td> Sigma-Aldrich公司</td><td> 435600 </td><td></td></tr><tr><td>胶原酶I型</td><td>西格玛</td><td> C0130 </td><td></td></tr></tbody></table>设备: <ol><li>一个荧光光谱仪（光子技术国际，新泽西州）:氙弧灯，电脑控制高速随机存取单色，试管基于TE-200倒置荧光发射单色器，尼康microscopE，的小号氟40x/1.30石油的目标，PMT探测器。 </li><li>激光扫描共聚焦显微镜（Radiance210​​0 BioRad公司）:453/477/488/514氩激光，氦氖激光543nm，尼康TE2000倒置显微镜，60X目标（不适用1.4，油）。 </li><li>一个立体显微镜放大功率&gt; 100（蔡司STEMI SV11 APO）。 </li></ol>

fluorescence-based measurement of store-operated calcium entry in live cells: from cultured cancer cell to skeletal muscle fiber

商店经营的Ca 2 +进入（SOCE），早期称为库容的Ca 2 +进入，是一个涌入细胞外Ca 2 +进入细胞，以补充枯竭的内质网（ER）或肌浆网（SR）的Ca 2 +商店严格监管机制1,2。由于钙离子是一种普遍存在的第二信使，它是不奇怪，SOCE多种细胞过程中起着重要的作用，包括增殖，凋亡，基因转录和运动。由于其广泛发生在几乎所有类型的细胞，包括上皮细胞和骨骼肌，这一途径已得到极大的兴趣3,4。然而，在不同类型的细胞和生理功能的SOCE特征的异质性仍然不明确5-7。 SOCE功能的通道特性可以发现，而大量的知识约氏体膜片钳研究基于荧光的细胞内钙离子的测量，因为它的方便和高通量筛选的可行性已经获得人生道路。本报告的目的是总结了一些基于荧光的方法来衡量SOCE在单层细胞，悬浮细胞和肌纤维5,8-10激活。最常使用这些荧光的方法是直接动态监测胞内Ca 2 +使用的F 340nm的发射波长（510 nm）的比例的Ca 2 +荧光浓度指示剂fura-2和F 380nm的比例。隔离开来单向SOCE活性细胞内Ca 2 +释放和Ca 2 +挤压，常用的Mn 2 +淬火法。锰2 +被称为是能够渗透到细胞通过SOCE，而这是防渗膜表面的挤压过程，或雌激素受体 ​​摄取钙+泵由于其非常高的亲和力机智ĤFURA-2。因此，外锰2 +进入细胞诱导的Fura-2荧光猝灭SOCE 9测量的活动。可以进行比率测量和Mn +2淬火检测细胞的人口模式，或在显微镜为基础的系统，以可视化的单细胞在试管基于分光光度计。单细胞测量的优点是可以选择使用GFP或RFP的记者，让转基因或突变的细胞研究，基因操作的单个细胞。 SOCE结构专门骨骼肌中的时空特征，可以实现皮肤的肌纤维，同时监测两个低亲和力钙荧光FLUO-5N，如肌纤维中的SR和具体的车厢有针对性地+指标Rhod 5N在横管9,11,12。

Watch this Scientific Journal Video about Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber at JoVE.com

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

店经营CA的程度<sup&gt; 2</sup使用荧光钙可以监视&gt;进入（SOCE）<sup&gt; 2</sup&gt;指标。锰<sup&gt; 2</sup&gt;等指标检测在培养细胞和骨骼肌纤维的SOCE淬火。允许的时间和空间分辨率SOCE机械剥皮的肌肉纤维的共焦成像的一种技术也被描述。

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 ...

fluorescence-based-measurement-store-operated-calcium-entry-live

Research

JoVE Journal

Biology

21.1K 次观看.  Robert Wood Johnson Medical School. 店经营CA的程度 2进入（SOCE） 2指标。锰 2等指标检测在培养细胞和骨骼肌纤维的SOCE淬火。允许的时间和空间分辨率SOCE机械剥皮的肌肉纤维的共焦成像的一种技术也被描述。

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

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.

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 ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.