JC-1线粒体膜电位检测试剂盒（JC-1 Mitochondrial Membrane Potential Assay Kit）是一种以JC-1 为荧光探针，快速灵敏地检测细胞、组织或纯化的线粒体膜电位变化的试剂盒，可以用于早期的细胞凋亡检测，也是用来检测细胞早期凋亡的常用方法。

JC-1 是一种广泛用于检测线粒体膜电位△Ψm 的理想荧光探针，JC-1染料以电势依赖性的方式积聚在线粒体内。正常线粒体内，JC-1聚集在线粒体基质中形成聚合物，聚合物发出强烈的红色荧光（Ex=585 nm, Em=590 nm）；不健康的线粒体由于膜电位的下降或丧失，JC-1只能以单体的形式存在于胞浆中，产生绿色荧光（Ex=514 nm, Em=529 nm）。JC-1不仅可用于定性检测，因颜色的变化可以非常直接的反映出线粒体膜电位的变化。也可以用于定量检测，因线粒体的去极化程度可以通过红/绿荧光强度的比例来衡量。观察时，只需使用常规的观察红色荧光和绿色荧光的设置即可。

线粒体膜电势的破坏是细胞凋亡早期发生的一个标志性事件。细胞受到凋亡诱导后线粒体膜电位的变化使得膜的通透性发生改变。膜通透性的增加，使得线粒体蛋白包括细胞色素C、Smac/DIABLO、HtrA2/OMI、核酸内切酶G（EndoG）、凋亡诱导因子（AIF）等从线粒体基质释放到细胞浆。细胞色素C的释放伴随膜电位的完全丧失，进而引发细胞凋亡酶的级联效应。

本试剂盒提供了CCCP 作为诱导线粒体膜电位下降的阳性对照。对于六孔板中的样品，本试剂盒共可以检测100 个样品；对于12 孔中的样品，本试剂盒共可以检测200 个样品。

冰袋（wet ice）运输。

-20℃避光保存，尽量避免反复冻融，有效期一年。超纯水和JC-1 染色缓冲液（5×）也可4℃保存。

Q：JC-1 探针和试剂盒有什么区别，客户做细胞实验用哪一款？

A:前者只是一个探针的粉末；后者是液体，又多了缓冲液，阳性对照和超纯水。两个都可以。

Q：JC-1 试剂盒检测的时候，客户要用流式检测，是要先把贴壁细胞消化下来再按照悬浮细胞处理吗？还是先染完色再消化下来去检测？

A:先消化下来制成细胞悬液再染色。

Q：阴性对照中线粒体都是正常的线粒体，为什么也可以观察到绿色的荧光，而且红色的也看不到细胞形态的原因是？

A：阴性对照可以拍到绿色可能是因为染色的时候加入的染料较多，从而使阴性对照可以排除绿色的荧光，可以适当的降低探针的浓度。绿色可以看到整个形态是因为单体（绿光）是散落在细胞质中的，而聚合体（红色）是结合到线粒体上的，有些细胞中线粒体分布是不均匀的，能否完整的看到细胞的形态是和线粒体的分布也是有一定的原因的。

Q：石蜡切片和冰冻切片可以做JC-1吗？

A：不可以，JC-1实验的样本必须是活细胞。

Q：JC-1孵育完成后不能及时检测，可以固定后再检测吗？

A：不可以，JC-1用于活细胞样本的检测，固定后细胞死亡；此外，染色后放置过久会导致荧光淬灭，建议染色30 min内完成检测。

Q：组织样本可不可以用JC-1检测凋亡？  

A：无法直接检测组织，可先将组织制备成单细胞悬液，再按照悬浮细胞的操作步骤来检测，需对制备方案进行摸索。

Q：JC-1工作液的配置中出现绿色沉淀应如何处理？  

A：取适量JC-1（200×），按照每50μL JC-1（200×）加入8 mL 超纯水的比例稀释JC-1，剧烈震荡充分溶解并混匀JC-1，然后再加入2 mL JC-1 染色缓冲液（5×），如溶液后存在绿色不溶沉淀可加入千分之一的Tween 20。

Q：该JC-1是否可用于分离纯化的完整线粒体膜电位的检测？JC-1的建议工作液如何配置？对应的纯化线粒体的量应为多少？加入JC-1后多久可以检测？  

A：可以用于纯化分离得到的完整线粒体的膜电位的检测。

JC-1的工作液及线粒体用量的比例为：0.9 mL JC-1染色工作液（1×）中加入0.1 mL总蛋白量为10-100 µg纯化的线粒体。

加入JC-1工作液3-5 min后即可进行检测。

但需留意对于冷冻的纯化线粒体不可用于进行膜电位的检测，因线粒体膜经冻融被破坏。

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