Why should I perform a compensation correction when using JC-10 in flow cytometry assays to quantitate mitochondrial membrane potential?

For example, Cell Meter^™ JC-10 Mitochondrion Membrane Potential Assay Kit (Cat# 22801) recommends that a compensation correction be performed.

JC-10 is an excellent fluorescent indicator utilized for monitoring mitochondrial membrane potential (∆Ψ_M). Compared to JC-1, JC-10 has improved water solubility making it less likely to precipitate in aqueous solutions. This allows for easier preparation and handling. Like JC-1, JC-10 selectively binds to mitochondria forming J-aggregates when the mitochondrion membrane is polarized. When excited at 490 nm, J-aggregates will emit at 570 nm. When the mitochondrion membrane is depolarized, JC-10 accumulation in mitochondria is low and a majority of JC-10 will be in their monomeric form. Excitation of JC-10 monomers at 490 nm will generate an emission at 525 nm. JC-10’s shift in emission is reversible and the signal ratio between 525 nm and 570 nm can be used to quantitate changes in ∆Ψ_M.

When monitoring ∆Ψ_M using a flow cytometer, the Argon Ion laser (488 nm) is used to detect the emission shift between 525 nm and 570 nm. We recommend using the FITC channel and PE-Texas Red channel to detect emission wavelengths at 530 nm and 615 nm, respectively. Because there is an emission overlap between J-monomers and J-aggregates, the signal from both can be detected in FITC and PE-Texas Red channels. This phenomenon is referred to as a bleed-over between FITC and PE-Texas Red channels. A compensation correction is performed to correct this spectral overlap. To do so, subtract the bleed-over J-monomer signal from the PE-Texas Red channel and subtract the bleed-over J-aggregates signal form the FITC channel. Without a compensation correction, the signal changes due to the changes in ∆Ψ_M might be compromised by the bleed-over signals producing a false negative result.