Assaying the effect of oligomycin on ΔψM using rhodamine 123 in quench mode in insulinoma cells results in internally inconsistent data.

(A) Whole cell fluorescence, (B) mitochondrial fluorescence obtained by high pass filtering and (C) fluorescence over the nuclei in rhodamine 123-loaded INS-1E cells following the additions of glucose (30 mM), oligomycin (2 μg/ml) and FCCP (1 μM). Data are mean±SE of n = 3 experiments. Note that the oligomycin-evoked drop in quench-mode whole-cell fluorescence (A) originates from the mitochondria (B) and not from the cytosol (approximated by rhodamine 123 fluorescence in the nucleosol) (C). (D) Representative images of INS-1E cells. From top to bottom: raw fluorescence, high pass filtered fluorescence to selectively transmit mitochondrial signal, and the temporal maximum intensity projection of the latter image series indicating that no mitochondria moved into the ROIs (dashed outlines) used to measure nuclear fluorescence (N). (E) Raw fluorescence, corresponding from top to bottom to baseline, glucose and oligomycin treatments. The red color indicates high intensity. (D-E) Scale Bar, 10 μm. (F) Mitochondrial fluorescence obtained by high pass filtering in INS-1 832/13 cells in the same condition as above. Representative trace of three experiments.