I am using JC-10 (Enzo life science) dye for quantify mitochondrial membrane potential in CRC with 10 uM final concentration. I treated cell with CCCP 10 uM for 20 min for a positive control. I took the reading for J-aggregate at Ex/Em: 490/525 nm and for monomeric JC-10 Ex/Em :490/590. However I could not get the appropriated 525/590 nm ratiometric decrease in MMP even in CCCP treated set. I also repeated the experiment applying JC-10 in serum free medium to stain the cell, and again I Couldn’t get the result. I have a doubt in my protocol of staining buffer. Many companies supplying JC-10 kit provide two types of assay buffer to stain the JC-10 in the cell. I could not find the information about the ingredients mixed in such assay buffers.

If you have any suggestion for me in staining buffer and in whole procedure. Please help

Thank you