CCCP英文全称为Carbonyl cyanide-m-chloro phenylhydrazone，又称碳酰氰基-对-氯苯腙，是一种离子载体、氧化磷酸化解偶联剂，可以抑制线粒体功能，还可抑制转运过程，延缓生长。JC-1是一种检测线粒体膜电位的理想荧光探针，在线粒体膜电位较高时JC-1聚集在线粒体的基质 (matrix)中形成聚合物，产生红色荧光；在线粒体膜电位较低时，JC-1不能聚集在线粒体的基质中，此时JC-1为单体，产生绿色荧光，通过JC-1从红色荧光到绿色荧光的转变可以很容易地检测到细胞膜电位的下降，这种转变也可作为细胞凋亡早期的一个检测指标。
CCCP溶液 (10 mmolL)是线粒体膜电位检测试剂盒 (JC-1法)的组成成分，该试剂盒是一种以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，CCCP作为诱导线粒体膜电位下降的阳性对照。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、JC-1染色液、磷酸盐缓冲液或PBS、蒸馏水或去离子水、细胞培养液
2、荧光分光光度计或流式细胞仪
操作步骤(仅供参考)：
1、加JC-1染色液：6孔板每孔加入1ml，其它培养器皿的用量酌情增减，对于细胞悬液每0.5-1.0×10⁶细胞需0.5ml JC-1染色液。
2、设置阳性对照：推荐CCCP (10mM)按照1:1000的比例加入到细胞培养液中，稀释至10μM，处理细胞20min，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20min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1-6×10⁵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20min。
c. 37℃孵育结束后， 4℃ 600g离心3-4min，沉淀细胞，弃上清。
d. 用PBS洗涤2次：加入1ml PBS重悬细胞，4℃ 600g离心3-4min，沉淀细胞，弃上清，重复一次。
e. 再用少量PBS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，应先收集细胞，重悬后参考悬浮细胞的检测方法。
a. 吸除6孔板培养液，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20min。
c. 37℃孵育结束后，吸除上清，用PBS洗涤2次。
d. 加入2ml细胞培养液，培养液中可以含有血清和酚红。
e. 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染色工作液再稀释5倍。
b. 0.9ml JC-1染色工作液中加入0.1ml总蛋白量为10-100μg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，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。
注意事项：
1、JC-1溶液应避免反复冻融。
2、装载完JC-1后用PBS洗涤时，尽量使PBS保持4℃左右，洗涤效果较好。
3、JC-1探针装载完并洗涤后尽量在30min内完成后续检测，在检测前需冰浴保存。
5、CCCP为线粒体电子传递链抑制剂，有一定毒性，请注意小心防护。
6、试剂开封后请尽快使用，以防影响后续实验效果。
7、试剂量较少，请先12000rpm离心1min后再使用。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Product Introduction
The full English name of CCCP is Carbonyl Cyanide-m-Chlorophenylhydrazone. It is an ionophore and oxidative phosphorylation uncoupler, which can inhibit mitochondrial function, suppress transport processes, and retard cell growth. JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 accumulates in the mitochondrial matrix to form polymers, emitting red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot accumulate in the mitochondrial matrix and exists as monomers, emitting green fluorescence. The decrease in mitochondrial membrane potential can be easily detected by the fluorescence shift of JC-1 from red to green, which can also serve as an indicator for the early stage of cell apoptosis.
CCCP Solution (10 mmol/L) is a component of the Mitochondrial Membrane Potential Assay Kit (JC-1 Method). This kit uses JC-1 as the fluorescent probe to rapidly and sensitively detect changes in mitochondrial membrane potential in cells, tissues, or purified mitochondria, and can be applied to early cell apoptosis detection. CCCP serves as a positive control to induce the decrease of mitochondrial membrane potential. This reagent is for research use only and not intended for clinical diagnosis or any other purposes.
Materials to Be Prepared by Users
1. JC-1 Staining Solution, Phosphate Buffered Saline (PBS), Distilled Water or Deionized Water, Cell Culture Medium
2. Fluorescence Spectrophotometer or Flow Cytometer
Operating Procedures (For Reference Only)
1. Adding JC-1 Staining Solution: Add 1 mL of the solution to each well of a 6-well plate. Adjust the dosage appropriately for other culture vessels. For cell suspension, 0.5 mL of JC-1 Staining Solution is required for every 0.5-1.0×10⁶ cells.
2. Setting Positive Control: It is recommended to dilute the 10 mM CCCP solution in cell culture medium at a ratio of 1:1000 to a final concentration of 10 μM, and treat the cells for 20 min. Then load JC-1 according to the method described below to detect mitochondrial membrane potential. For most cell types, treatment with 10 μM CCCP for 20 min usually leads to a complete loss of mitochondrial membrane potential, and the cells should show green fluorescence after JC-1 staining. In contrast, normal cells should exhibit red fluorescence after JC-1 staining. For specific cell types, the effective concentration and treatment time of CCCP may vary, which should be determined by referring to relevant literature.
3. For Suspension Cells:
a. Take 1-6×10⁵ cells and resuspend them in 0.5 mL of cell culture medium, which may contain serum and phenol red.
b. Add 0.5 mL of JC-1 Staining Working Solution, invert the tube several times to mix well, and incubate the cells at 37℃ for 20 min in a cell incubator.
c. After incubation at 37℃, centrifuge the cells at 600×g and 4℃ for 3-4 min to pellet the cells, then discard the supernatant.
d. Wash the cells twice with PBS: Add 1 mL of PBS to resuspend the cells, centrifuge at 600×g and 4℃ for 3-4 min, discard the supernatant, and repeat this step once.
e. Resuspend the cells in a small amount of PBS, then observe them under a fluorescence microscope or laser confocal microscope. Detection can also be performed using a fluorescence spectrophotometer or analysis with a flow cytometer.
4. For Adherent Cells:
Note: For adherent cells, if detection by fluorescence spectrophotometer or flow cytometer is desired, the cells should be harvested first, resuspended, and then detected by referring to the method for suspension cells.
a. Aspirate the culture medium from the 6-well plate. If necessary according to the specific experiment, wash the cells once with PBS or other appropriate solution, then add 1 mL of cell culture medium, which may contain serum and phenol red.
b. Add 1 mL of JC-1 Staining Working Solution, mix thoroughly, and incubate the cells at 37℃ for 20 min in a cell incubator.
c. After incubation at 37℃, aspirate the supernatant and wash the cells twice with PBS.
d. Add 2 mL of cell culture medium, which may contain serum and phenol red.
e. Observe the cells under a fluorescence microscope or laser confocal microscope.
5. For Purified Mitochondria:
a. Further dilute the prepared JC-1 Staining Working Solution 5-fold.
b. Add 0.1 mL of purified mitochondria with a total protein amount of 10-100 μg to 0.9 mL of the diluted JC-1 Staining Working Solution.
c. Detection with Fluorescence Spectrophotometer or Fluorescence Microplate Reader: Mix well and directly perform time-scan detection using a fluorescence spectrophotometer with the excitation wavelength set at 485 nm and emission wavelength at 590 nm. If the fluorescence microplate reader cannot set the excitation wavelength to 485 nm, the excitation wavelength can be set within the range of 475-520 nm. Alternatively, the wavelength settings in Step 6 below can be referred to for fluorescence detection.
d. Observation with Fluorescence Microscope or Laser Confocal Microscope: Follow the method described in Step 6 below.
6. Fluorescence Observation and Result Analysis:
For detecting JC-1 monomers, set the excitation wavelength at 490 nm and emission wavelength at 530 nm. For detecting JC-1 polymers, set the excitation wavelength at 525 nm and emission wavelength at 590 nm. Note: It is not necessary to set the excitation and emission wavelengths at their maximum values for fluorescence measurement here. If using a fluorescence microscope for observation, the settings for detecting JC-1 monomers can refer to those used for observing other green fluorescent substances, such as GFP or FITC. The settings for detecting JC-1 polymers can refer to those used for observing other red fluorescent substances, such as Propidium Iodide (PI) or Cy3. The appearance of green fluorescence indicates a decrease in mitochondrial membrane potential, suggesting that the cells are likely in the early stage of apoptosis. The appearance of red fluorescence indicates that the mitochondrial membrane potential is relatively normal and the cell status is good.
Precautions
1. Avoid repeated freeze-thaw cycles of the JC-1 solution.
2. When washing the cells with PBS after JC-1 loading, keep the PBS at around 4℃ as much as possible to achieve a better washing effect.
3. Try to complete the subsequent detection within 30 min after JC-1 probe loading and washing. Store the samples on ice before detection.
4. CCCP is an inhibitor of the mitochondrial electron transport chain and has certain toxicity. Please handle it with care and take appropriate protective measures.
5. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
6. The volume of the reagent is small. Please centrifuge it at 12,000 rpm for 1 min before use.
7. For your safety and health, please wear a lab coat and disposable gloves during operation.
ZJ26F0636281
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