Uncompensated Polychromatic Analysis of Mitochondrial Membrane Potential Using JC-1 and Multilaser Excitation

The lipophilic cation JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨ_m). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, ΔΨ_m along with other biological parameters, such as apoptosis and the production of reactive oxygen species. © 2015 by John Wiley & Sons, Inc.