Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). Labeling of Cells: 1. Culture cells in 6-, 12- , 24-, or 96-well plates at a density of 5× 10 5 cells/mL. Incubate the cells according to your normal protocol. 2. Ensure that the JC-1 and DMSO has equilibrated to room temperature, and then prepare a 200 μM stock solution by dissolving the contents of one vial in 230 μL of the DMSO provided. 3. For the control tube, allow the vial of CCCP has come to room temperature, add 1 μL of CCCP (50 mM). Incubate cells at 37°C for 5 minutes. 4. Add 10 μL JC-1 (200 μM) per well to make the final concentration at 2 μM. Incubate cells at 37°C, 5% CO 2 , for 15-20 minutes. If additional labeling followed, for example with an annexin V, begin with step 2.a. If not, proceed with step 1.e. 5. After incubation, centrifuge cells for 3-4 minutes at 400× g at 4°C, carefully aspirate the supernant. 6. Wash cells twice with PBS (1×): add 2 mL PBS (1×) to suspend cells and vortex to mix thoroughly. Centrifuge cells for 3-4 minutes at 400× g at 4°C, carefully aspirate the supernant. 7. Add 500 μL PBS (1×) to suspend cells. Analyze sample on a flow cytometer, fluorescence microscopy, or fluorescence microplate reader.

JC-1 (CBIC2) 是荧光亲脂性羰花青染料，用于测量线粒体膜电位。线粒体膜电位较高时， JC-1 在基质中汇聚形成聚合物 (J-aggregates)，可以产生红色荧光 (Ex/Em=585/590 nm)；线粒体膜电位较低时，JC-1 不能聚集在线粒体基质中，以单体形式存在产生绿色荧光 (Ex/Em=510/527 nm)。