Hi,

I used the JC10 kit (MAK159) to measure mitochondrial membrane potential in neuronal cells. For positive controls, we used FCCP at 10 µM and oligomycin at 3 µg/mL, with the former to induce hypopolarization, and the latter intended to induce hyperpolarization.

However, the results showed unexpected hyperpolarization with FCCP, as the ratio and percentage were significantly higher compared to both control untreated cells.

No issues with oligomycin. It gave 107%; which indicates hyperpolarization as expected.

To prepare FCCP, I diluted a 50 mM stock (dissolved in DMSO) to 1 mM using PBS, and then further diluted it in the kit’s Buffer A to a final concentration of 10 µM/well.

Could anyone advise why these unusual results occurred? The normalized ratio relative to the control was 160%, which is unexpectedly high, especially since FCCP should induce depolarization, not hyperpolarization.

Thank you in advance