JC-1线粒体膜电位荧光探针

产品简介

JC-1线粒体膜电位荧光探针在通过流式细胞术检测线粒体膜电位中广泛使用。它能够选择性的进入线粒体中，并且由于膜电位的增加（大约超过80-100 mV）其颜色会发生从绿色到橙色的可逆变化。这种特性是由于JC-1聚合物的可逆结构，在膜极化条件下，它的发射光由530nm(J-单体发射光)转移到590nm (JC-1聚合体发射光)。当在490nm处激发时，JC-1发生从绿色到橙绿色的可逆改变，因为线粒体膜极化的更大。这两种颜色都可以被流式细胞仪上装好的一般滤光器检测到，因此绿色发射光可以通过荧光通道1（FL1）进行分析，橙绿色发射光可以通过荧光通道2（FL2）进行分析。JC-1主要的优点是，它既可以进行定性检测，通过其发射荧光的颜色从绿色到橙色的改变；也可以进行定量检测，通过对两个检测通道FL1和FL2单一色彩的荧光强度进行检测；它也可以用于荧光成像分析。We have developed a protocol to use it in fluorescence microplate platform.。虽然JC-1广泛地用于许多实验中，但是其水溶性差，也导致它很难在一些实验中使用。我们的JC-10的水溶性比JC-1要高，并且在许多细胞系中有着超好的性能表现；有趣地是，JC-10的性能表现对细胞系极具依赖性。

储存条件：

-20℃

操作说明（仅供参考）

1. Prepare JC-1 working solution:

1. Prepare a 2 to 10 mM stock solution of JC-1 in high-quality, anhydrous DMSO. The stock solution should be used promptly; any remaining solution should be aliquoted and frozen at<-20°C;.
   Note: Avoid repeated freeze-thaw cycles, and protect from light.
2. Prepare a 1X JC-1 working solution: On the day of the experiment, either dissolve JC-1 solid in DMSO or thaw an aliquot of the JC-1 stock solution to room temperature. Prepare a 10 to 30 µM 1X JC-1 working solution in Hanks and 20 mM Hepes buffer (HHBS) or buffer of your choice, pH 7 with 0.02% Pluronic® F-127. Mix them well by votexing.
   Note: JC-1is not water soluble, so it intends to aggregate in solution. It is recommended to filter the JC-1 working solution before loading it into the cells.

2. Run JC-1 assay with a fluorescence microplate reader:

1. Treat cells with test compounds for a desired period of time (For example, Jurkat cells can be treated with camptothecin for 4-6 hours) to induce apoptosis. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
2. Add 100 µL/well/96-well plate or 25 µL/well/384-well plate of JC-1 working solution (from Step 1.b) into the cell plate.
3. Incubate the JC-1 loading plate in a 37°C, 5% CO₂ incubator for 15-60 min.
   Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
4. Remove the JC-1 working solution from the plate, wash the cells with HHBS or buffer of your choice. Add 100 µL/well/96-well plate or 25 µL/well/384-well plate of HHBS back to the cell plate.
5. Monitor the fluorescence change atEx/Em = 490/525 nm and 490/590 nm for ratio analysis.

3. Run JC-1 assay with a fluorescence microscope or a flow cytometer:

1. Treat cells with test compounds for a desired period of time (For example, Jurkat cells can be treated with camptothecin for 4-6 hours) to induce apoptosis.
2. Centrifuge the cells to get 1-5 x 105cells per tube.
3. Resuspend cells in 500 µL of JC-1 working solution (from Step 1.b).
4. Incubate at room temperature or 37° C for 10 to 30 min, protected from light.
5. Wash the cells with HHBS or buffer of your choice. Resuspend cells in 500 µL of HHBS to get 1-5×10⁵ cells per tube.
6. Monitor the fluorescence change at Ex/Em = 490/525 nm and 490/590 nm with a fluorescence microscope (using FITC and TRITC filters) or a flow cytometer (using FL1 and FL2 channels).