使用说明：
1. JC-1染色工作液的配制：
    六孔板每孔所需JC-1染色工作液的量为1ml，其它培养器皿的JC-1染色工作液的用量以此类推；对于
    细胞悬液每50-100万细胞需0.5ml JC-1染色工作液。取适量JC-1(200X)，按照每50微升JC-1(200X)
    加入8ml超纯水的比例稀释JC-1。剧烈Vortex充分溶解并混匀JC-1。然后再加入2ml JC-1染色缓冲液
    (5X)，混匀后即为JC-1染色工作液。
2. 阳性对照的设置：
    把试剂盒中提供的CCCP(10mM)推荐按照1:1000的比例加入到细胞培养液中，稀释至10μM，处理细
    胞20分钟。随后按照下述方法装载JC-1，进行线粒体膜电位的检测。对于大多数细胞，通常10μM
    CCCP处理20分钟后线粒体的膜电位会完全丧失，JC-1染色后观察应呈绿色荧光；而正常的细胞经
    JC-1染色后应显示红色荧光。对于特定的细胞，CCCP的作用浓度和作用时间可能有所不同，需自行
    参考相关文献资料确定。
3. 对于悬浮细胞：
    a. 取10-60万细胞，重悬于0.5ml细胞培养液中，细胞培养液中可以含血清和酚红。
    b. 加入0.5ml JC-1染色工作液，颠倒数次混匀。细胞培养箱中37℃孵育20分钟。
    c. 在孵育期间，按照每1ml JC-1染色缓冲液(5X)加入4ml蒸馏水的比例，配制适量的JC-1染色缓冲
       液(1X)，并放置于冰浴。
    d. 37℃孵育结束后， 600g 4℃离心3-4分钟，沉淀细胞。弃上清，注意尽量不要吸除细胞。
    e. 用JC-1染色缓冲液(1X)洗涤2次：加入1ml JC-1染色缓冲液(1X)重悬细胞，600g 4℃离心3-4分
       钟，沉淀细胞，弃上清。再加入1ml JC-1染色缓冲液(1X)重悬细胞，600g 4℃离心3-4分钟，沉
       淀细胞，弃上清。
    f. 再用适量JC-1染色缓冲液(1X)重悬后，用荧光显微镜或激光共聚焦显微镜观察，也可以用荧光分
       光光度计检测或流式细胞仪分析。
4. 对于贴壁细胞：
    注意：对于贴壁细胞，如果希望采用荧光分光光度计或流式细胞仪检测，可以先收集细胞，重悬后
    参考悬浮细胞的检测方法。
    a. 对于六孔板的一个孔，吸除培养液，根据具体实验如有必要可以用PBS或其它适当溶液洗涤细胞
       一次，加入1ml细胞培养液。细胞培养液中可以含有血清和酚红。
    b. 加入1ml JC-1染色工作液，充分混匀。细胞培养箱中37℃孵育20分钟。
    c. 在孵育期间，按照每1ml JC-1染色缓冲液(5X)加入4ml蒸馏水的比例，配制适量的JC-1染色缓冲
       液(1X)，并放置于冰浴。
    d. 37℃孵育结束后， 吸除上清，用JC-1染色缓冲液(1X)洗涤2次。
    e. 加入2ml细胞培养液，培养液中可以含有血清和酚红。
    f. 荧光显微镜或激光共聚焦显微镜下观察。
5. 对于纯化的线粒体：
    a. 把配制好的JC-1染色工作液再用JC-1染色缓冲液(1X)稀释5倍。
    b. 0.9ml 5倍稀释的JC-1染色工作液中加入0.1ml总蛋白量为10-100微克纯化的线粒体。
    c. 用荧光分光光度计或荧光酶标仪检测：混匀后直接用荧光分光光度计进行时间扫描(time scan)，
       激发波长为485nm，发射波长为590nm。如果使用荧光酶标仪，激发波长不能设置为485nm
       时，可以在475-520nm范围内设置激发波长。另外，也可以参考下面步骤6中的波长设置进行荧
       光检测。
    d. 用荧光显微镜或激光共聚焦显微镜观察：方法同下面的步骤6。
6. 荧光观测和结果分析：
    检测JC-1单体时可以把激发光设置为490nm，发射光设置为530nm；检测JC-1聚合物时，可以把激发
    光设置为525nm，发射光设置为590nm。注意：此处测定荧光时不必把激发光和发射光设置在最大
    激发波长和最大发射波长。如使用荧光显微镜观察，检测JC-1单体时可以参考观察其它绿色荧光时
    的设置，如观察GFP或FITC时的设置；检测JC-1聚合物时可以参考观察其它红色荧光，如碘化丙啶
    或Cy3时的设置。出现绿色荧光说明线粒体膜电位下降，并且该细胞很可能处于细胞凋亡早期。出
    现红色荧光说明线粒体膜电位比较正常，细胞的状态也比较正常。

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