Figure 3.

Characterization of MB-containing ICB permeability. To measure the permeability of MB-containing ICB, HeLa cells expressing MKLP1-EGFP were used. The pipette filled with the dye was attached to the cell-1. After opening patch by electrical pulse, the dye diffused to the cell-1 followed by dye transfer through the ICB to the cell-2. Kinetics of dye accumulation in both cells was measured by time-lapse imaging. Cells were exposed to low-intensity light for ~0.5 s every 1 min to minimize dye bleaching. During the entire experiment, tested cells were maintained in continuous external perfusion through a rectangular glass capillary situated in the proximity to the cells in order to prevent possible external contamination of cells with the dye due to its leak from the patch pipette. Representative images from at least three independent experiments for each fluorescent dye are shown. MB-containing ICBs were permeable to negatively charged dyes AF350 (A, 1 mM), LY (B, 1 mM), and AF546/10,000 (C, 0.1 mM) and impermeable to positively charged dyes DAPI (D, 0.1 mM) and Hoechst-33342 (E, 0.05 mM).