JC-1 Mitochondrial Membrane Potential Dye

JC-1 is a green-orange fluorescent dye used to assay mitochondrial membrane potential for studying cell health in flow cytometry applications.

Mechanism and principle

JC-1 (5,5,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide) is a cationic dye that selectively labels mitochondria in live cells. In response to increases in mitochondrial transmembrane potential (ΔΨ_M), from approximately 80 to 100 mV, the dye changes color from green to orange. The mechanism for this is the reversible formation of JC-1 aggregates upon membrane polarization; high membrane potential results in JC-1 monomer aggregation into J aggregates. These J aggregates exhibit orange fluorescence, while JC-1 monomers fluoresce green. By monitoring the intensity and ratios of green/orange fluorescences, JC-1 staining can be used as an assay for mitochondrial membrane potential.

Excitation and emission

Under low mitochondrial membrane potential conditions, JC-1 molecules exist largely as monomers. These have an absorption peak of 510 nm and an emission peak of 527 nm. Under polarized conditions (high ΔΨ_M), J aggregates form, which have an absorption peak of 585 nm and an emission peak of 590 nm. Both JC-1 monomers (green form) and J aggregates (orange form) can be excited by a 488 nm argon laser. The JC-1 monomer green emission can be detected using the FL1 channel of a flow cytometer, while the J aggregate orange emission can be detected using the FL2 channel.

Form	Mitochondria Membrane Potential	Color	Absorption (nm)	Emission (nm)	Emission Channel
JC-1 monomer	Low	Green	510	527	FL1
J aggregate	High	Orange	585	590	FL2

JC-1 Alternatives

Although JC-1 is widely used in many labs, one problem it suffers from is poor water solubility. An alternative version, which addresses this issue, is JC-10.

Sivandzade, F., Bhalerao, A., & Cucullo, L. (2019). Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe. Bio-protocol, 9(1), e3128. https://doi.org/10.21769/BioProtoc.3128