APPENDIX A: Ambiguities observed with spontaneous FM 1-43 labeling in epithelial cells

Several labs have measured the spontaneous and stimulated increases in FM 1-43 fluorescence combined with simultaneous capacitance measurements to confirm the method’s viability (1-4). In these tests, the capacitance of single cells was measured using whole-cell patch clamping. By design, the corresponding fluorescence changes were also measured on single cells, either by monitoring changes in the mean fluorescence intensity (MFI) in an ROI encompassing a portion of the labeled plasma membrane and cytoplasm (“edge fluorescence”, (4)), or the entire cell (“total cellular fluorescence”, (2, 3, 5)). At least one lab measured the MFI of FM 1-43 stained polarized monolayers, and found reasonable agreement with separate measurements performed on single cells (2). Our studies measuring the capacitance of single cells using whole cell patch clamping (6) and intact monolayers using impedance analysis (7, 8) had already revealed significant differences in the apparent exocytic response of these two cell configurations. The exocytic response of cell clusters could not be measured with electrophysiology: while it is possible to achieve the whole cell patch configuration on a single cell in a cluster, clearly only the attached cell is directly clamped. Attempts to measure the capacitance of these attached cells consistently reported significantly larger membrane capacitances (Fig. A1) than seen in individual cells of similar (apparent) size. This may be due to coupling through gap junctions, and/or problems with space clamp rendering the 3 parameter model (9) used to fit the exponential transients inadequate. The cell cluster model is frequently used in fluorescence assays and may provide a bridge between the single cell and monolayer configurations, hence we examined whether dynamic changes in FM 1-43 fluorescence could be reliably measured.