Measurement of ΔψM in rodent primary β-cells.

(A) Wide-field fluorescence view field of a dispersed rat islet cell culture loaded with PMPI (left; green) and TMRM (right; red). a) 3 mM glucose baseline; b) 16 mM glucose; c) complete depolarization at the end of the experiment, corresponding to the time courses shown in B-G; d) insulin immunofluorescence (red) and Hoechst 33342 nuclear staining (blue) in the same view field. (B-C) Fluorescence intensity time courses of the potentiometric probes. (D-E) Time courses of calibrated potentials corresponding to B-C were calculated using VF = 0.078 and aR’ = 0.296 and biophysical constants of the probes as previously published [21]. Data are mean±SE of n = 34 cells individually calibrated and pooled in five view fields in a single well, and representative of 3 independent experiments. Potentials were calibrated in single or aggregates of insulin-positive cells as indicated by regions of interests in Aa. (F-G) Calibrated potentials in 3 (groups of) cells indicated in Ab by arrows. Error bars indicate the predicted SE of the calibration calculated by propagation of the errors in determinations of calibration parameters. (H) Calculation of parameters of ΔψP calibration using linear regression on normalized fluorescence intensities and extracellular [K+] ([K+]ec) values during “calibration steps” shown in B. (I) Calculation of parameters of ΔψM calibration using linear regression on normalized fluorescence intensities of TMRM decay. H and I correspond to cells indicated in Aa, F and G. Normalization was performed as previously published [21].