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JC-10是一种检测线粒体膜电位（mitochondrial membrane potential）的理想荧光探针，能够快速灵敏地反映细胞、组织或者纯化的线粒体膜电位的变化。
在线粒体膜电位较高时，JC-10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-10不能聚集在线粒体的基质中，此时JC-10为单体，产生绿色荧光。这样就可以通过荧光颜色的转变来检测线粒体膜电位的变化，常用红绿荧光的相对比例来衡量线粒体去极化的比例。线粒体膜电位的下降是细胞凋亡早期的一个标志性事件。通过JC-10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。JC-10单体的最大激发波长为490nm，最大发射波长为525nm。JC-10聚合物（J-aggregates）的最大激发波长为540nm，最大发射波长为590nm，荧光分光光度计检测红/绿色荧光强度的比率用于测定线粒体膜电位（MMP）。实际观察时使用常规的观察红色荧光和绿色荧光的装置即可。
线粒体膜电位检测试剂盒（JC-10）是一种以JC-10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。该试剂盒仅用于科研领域，不宜用于临床诊断或其他用途。
操作步骤（仅供参考）
1、配制JC-10染色工作液
取适量JC-10 Stain (200×)，按照每50μL JC-10 Stain (200×) 加入8mL ddH₂O的比例稀释JC-10，剧烈Vortex充分溶解并混匀JC-10 Stain。然后再加入2mL JC-10 Buffer (5×)，混匀后即为JC-10染色工作液。6孔板每孔所需JC-10染色工作液的量为1mL，其它培养器皿的JC-10染色工作液的用量以此类推；对于细胞悬液每0.5～1.0×10⁶细胞需0.5mL JC-10染色工作液。
2、设置阳性对照
推荐CCCP (1mM) 按照1:100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-10，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-10染色后观察应呈绿色荧光；而正常的细胞经JC-10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
(1) 取1×10⁵～6×10⁵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。
(2) 加入0.5mL JC-10染色工作液，颠倒数次混匀。细胞培养箱中37℃孵育20min。
(3) 在孵育期间，按照每1mL JC-10 Buffer (5×) 加入4mL蒸馏水的比例，配制适量的JC-10 Buffer (1×)，并放置于冰浴。
(4) 37℃孵育结束后，4℃ 600g离心3～4min，沉淀细胞。弃上清，注意尽量不要吸除细胞。
(5) 用JC-10 Buffer (1×) 洗涤2次：加入1mL JC-10 Buffer (1×) 重悬细胞，4℃ 600g离心3～4min，沉淀细胞，弃上清。再加入1mL JC-10 Buffer (1×) 重悬细胞，4℃ 600g离心3～4min，沉淀细胞，弃上清。
(6) 再用少量JC-10 Buffer (1×) 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。
(1) 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。
(2) 加入1mL JC-10染色工作液，充分混匀。细胞培养箱中37℃孵育20min。
(3) 在孵育期间，按照每1mL JC-10 Buffer (5×) 加入4mL蒸馏水的比例，配制适量的JC-10 Buffer (1×)，并放置于冰浴。
(4) 37℃孵育结束后，吸除上清，用JC-10 Buffer (1×) 洗涤2次。
(5) 加入2mL细胞培养液，培养液中可以含有血清和酚红。
(6) 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
(1) 把配制好的JC-10染色工作液再用JC-10 Buffer (1×) 稀释5倍。
(2) 0.9mL JC-10染色工作液中加入0.1mL总蛋白量为10～100μg纯化的线粒体。
(3) 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。
(4) 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-10单体时可以把激发光设置为490nm，发射光设置为530nm；检测JC-10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-10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-10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-10 Stain (200×) 应完全溶解混匀后使用，但应避免反复冻融。必须先把JC-10用ddH₂O充分溶解混匀后，才可加入JC-10 Buffer (1×)。不可先配制JC-10 Buffer (1×) 再加入JC-10 Stain (200×)，否则导致JC-10很难充分溶解，严重影响后续的检测。
2、对于6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。
3、装载完JC-10后用JC-10 Buffer (1×) 洗涤时，尽量使JC-10 Buffer (1×) 保持4℃左右，此时的洗涤效果较好。
4、JC-10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、勿把JC-10 Buffer (5×) 全部配制成1×，因为操作过程中需直接使用JC-10 Buffer (5×)。
6、如JC-10 Buffer (5×) 中有沉淀，必须全部溶解后才能使用，为促进溶解可以在37℃加热。
7、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。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
JC-10 is an ideal fluorescent probe for detecting mitochondrial membrane potential (ΔΨm). It can rapidly and sensitively reflect changes in the membrane potential of cells, tissues, or purified mitochondria.
When the mitochondrial membrane potential is high, JC-10 accumulates in the mitochondrial matrix and forms aggregates (J-aggregates), which emit red fluorescence. When the mitochondrial membrane potential is low, JC-10 cannot accumulate in the matrix and remains in its monomeric form, emitting green fluorescence. Thus, changes in mitochondrial membrane potential can be detected by the shift in fluorescence color. The relative ratio of red to green fluorescence is commonly used to assess the proportion of mitochondrial depolarization. A decrease in mitochondrial membrane potential is an early hallmark event of apoptosis. The transition of JC-10 fluorescence from red to green easily indicates this depolarization and serves as an early indicator of apoptosis. JC-10 monomers have maximum excitation and emission wavelengths of 490 nm and 525 nm, respectively. JC-10 aggregates (J-aggregates) have maximum excitation and emission wavelengths of 540 nm and 590 nm, respectively. The red/green fluorescence intensity ratio, measured by a fluorescence spectrophotometer, is used to determine the mitochondrial membrane potential (MMP). For observation, standard setups for red and green fluorescence are sufficient.
The Mitochondrial Membrane Potential Assay Kit (JC-10) uses JC-10 as the fluorescent probe to rapidly and sensitively detect changes in the membrane potential of cells, tissues, or purified mitochondria. It can be used for early apoptosis detection. CCCP is included as a positive control for inducing mitochondrial membrane potential depolarization. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.
Operating Steps (for reference only)
1. Preparation of JC-10 Staining Working Solution
Take an appropriate amount of JC-10 Stain (200×). Dilute JC-10 by adding 8 mL of ddH₂O per 50 μL of JC-10 Stain (200×). Vortex vigorously to dissolve and mix the JC-10 Stain completely. Then, add 2 mL of JC-10 Buffer (5×) and mix well. This is the JC-10 Staining Working Solution. For a 6-well plate, 1 mL of working solution is needed per well.Scale accordingly for other culture vessels. For cell suspensions, use 0.5 mL of working solution per 0.5–1.0 × 10⁶ cells.
2. Setting Up a Positive Control
It is recommended to add CCCP (1 mM) to the cell culture medium at a 1:100 ratio to achieve a final concentration of 10 μM. Treat cells for 20 minutes. Then, load JC-10 as described below to detect mitochondrial membrane potential. For most cells, 10 μM CCCP treatment for 20 minutes typically causes complete loss of mitochondrial membrane potential, resulting in green fluorescence after JC-10 staining. Normal cells stained with JC-10 should show red fluorescence. For specific cell types, the optimal CCCP concentration and treatment time may vary; please refer to relevant literature.
3. For Suspension Cells
(1) Take 1×10⁵ to 6×10⁵ cells and resuspend them in 0.5 mL of cell culture medium (which may contain serum and phenol red).
(2) Add 0.5 mL of JC-10 Staining Working Solution and mix by inverting several times. Incubate at 37°C in a cell culture incubator for 20 minutes.
(3) During incubation, prepare an adequate amount of JC-10 Buffer (1×) by adding 4 mL of distilled water to every 1 mL of JC-10 Buffer (5×). Keep it on ice.
(4) After incubation, centrifuge at 4°C, 600 g for 3–4 minutes to pellet cells. Carefully aspirate the supernatant without disturbing the pellet.
(5) Wash cells twice with JC-10 Buffer (1×): Resuspend the pellet in 1 mL of JC-10 Buffer (1×), centrifuge at 4°C, 600 g for 3–4 minutes, and aspirate the supernatant. Repeat once.
(6) Resuspend the cells in a small volume of JC-10 Buffer (1×) and observe under a fluorescence microscope or confocal laser scanning microscope. Alternatively, analyze using a fluorescence spectrophotometer or flow cytometer.
4. For Adherent Cells
Note: For adherent cells intended for analysis by fluorescence spectrophotometer or flow cytometry, harvest and resuspend the cells first, then follow the protocol for suspension cells.
(1) Aspirate the medium from the 6-well plate. If necessary, wash cells once with PBS or an appropriate solution. Add 1 mL of cell culture medium (which may contain serum and phenol red).
(2) Add 1 mL of JC-10 Staining Working Solution and mix well. Incubate at 37°C in a cell culture incubator for 20 minutes.
(3) During incubation, prepare JC-10 Buffer (1×) as described in step 3.3 and keep it on ice.
(4) After incubation, aspirate the supernatant and wash the cells twice with JC-10 Buffer (1×).
(5) Add 2 mL of cell culture medium (which may contain serum and phenol red).
(6) Observe under a fluorescence microscope or confocal laser scanning microscope.
5. For Purified Mitochondria
(1) Further dilute the prepared JC-10 Staining Working Solution 5-fold with JC-10 Buffer (1×).
(2) Add 0.1 mL of purified mitochondria (total protein 10–100 μg) to 0.9 mL of the diluted JC-10 Staining Working Solution.
(3) Detection by fluorescence spectrophotometer or fluorescence microplate reader: Mix and immediately perform a time scan using a fluorescence spectrophotometer (excitation: 485 nm, emission: 590 nm). If using a microplate reader where 485 nm is unavailable, set the excitation wavelength between 475–520 nm. Alternatively, follow the wavelength settings in step 6 below.
(4) Observation by fluorescence or confocal microscope: Follow the method in step 6 below.
6. Fluorescence Observation and Analysis
For JC-10 monomers, set excitation at 490 nm and emission at 530 nm. For JC-10 aggregates (J-aggregates), set excitation at 525 nm and emission at 590 nm.
Note: It is not necessary to use the exact maximum excitation/emission wavelengths. For fluorescence microscopy, settings for JC-10 monomers can be similar to those for GFP or FITC (green fluorescence). Settings for JC-10 aggregates can be similar to those for Propidium Iodide or Cy3 (red fluorescence). Green fluorescence indicates decreased mitochondrial membrane potential, suggesting the cell is likely in early apoptosis. Red fluorescence indicates normal mitochondrial membrane potential and cell state.
Precautions
JC-10 Stain (200×) should be completely dissolved and mixed before use. Avoid repeated freeze-thaw cycles. JC-10 must be fully dissolved in ddH₂O before adding JC-10 Buffer (5×). Do not add JC-10 Stain to pre-prepared JC-10 Buffer (1×), as this will prevent proper dissolution and severely affect detection.
This kit is sufficient for 100 samples in a 6-well plate or 200 samples in a 12-well plate.
When washing with JC-10 Buffer (1×) after JC-10 loading, keep the buffer at approximately 4°C for better washing efficiency.
Complete detection within 30 minutes after JC-10 loading and washing. Keep samples on ice before detection.
Do not prepare all JC-10 Buffer (5×) into 1× buffer, as the 5× concentrate is needed during the procedure.
If precipitate forms in JC-10 Buffer (5×), ensure it is completely dissolved before use. Heating at 37°C may aid dissolution.
CCCP is a mitochondrial electron transport chain inhibitor and is toxic. Handle with care and appropriate protection.
For your safety and health, please wear a lab coat and disposable gloves during operation.
| M1509036 | Component | 100T | Storage |
| M1509036A | JC-10 Stain (200×) | 100 μL×5 | -20℃. Store in the dark. |
| M1509036B | JC-10 Buffer (5×) | 80 mL | 2-8℃. |
| M1509036C | CCCP (1mM) | 200 μL | -20℃. |
| M1509036D | ddH₂O | 90 mL | RT. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0232049 |
分析证书 | M1509036 |
| ZJ26F0232065 |
分析证书 | M1509036 |
| ZJ26F0232066 |
分析证书 | M1509036 |
| ZJ26F0232067 |
分析证书 | M1509036 |
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