(a) SEM of the sucrose-gap chip. Three inlet channels are connected to a sensing channel that is defined by two pair of electrodes on either side. Downstream two outlet channels split the solution into separate reservoirs. (b) Numerical simulation of current distribution, showing that the electrical field is confined in the sensing chamber (comsol/multiphysics). (c) A cross-section view of the sensing channel along the dashed line in (a), showing how a non-conductive sucrose gap is created by the middle stream so the electrical pathway is only through the confluent cells. (d) Cross-section view of current distribution in the flow chamber containing confluent cells. The conductivity of cytoplasm, cell membrane, and gap junction was 0.1, 0.2 × 10−6, and 1 × 10−4 S/m, respectively. The parameters are from Refs. 38 and 39. It shows that the current lines are concentrated through the cell sheet in the gap region.
Calibration of microfluidic chip. (a) Changes in resistance across the gap in empty chamber when the central channel was repeatedly perfused with sucrose solution with and without 2-APB (200 μM), showing that a repeatable gap resistance can be attained with multiple perfusions and that the addition of drug does not alter the gap resistance. (b) A stable gap resistance is maintained during solution exchange in central stream. (c) Changes in resistance across the gap when the central channel was perfused with sucrose solution in the presence of confluent cells. The ground resistance in (a) and (c) is the chamber resistance without sucrose gap.
Effect of 2-APB on electrical coupling of gap junction channels in NRK cells. (a) The central stream was switched between sucrose and sucrose with 2-APB (200 μM) repeatedly (indicated by arrows), showing that the drug reversibly blocks the channels. (b) A control experiment for the effect of DMSO used as the 2-APB vehicle, showing that the vehicle did not block the channels. The central stream was perfused alternately with sucrose + DMSO and sucrose. (c) and (d) Images of the cells before and after the experiment at the times indicated by the red arrows in (a), showing that the cells remain attached during the measurement. The scale bar represents 100 μm.
Dose dependent 2-APB inhibition. (a) Representative changes in resistance measured in the presence of 50 μM (black), 100 μM (red), 200 μM (green), and 400 μM (blue) 2-APB. The change in resistance (ΔR) was normalized to the baseline resistance (R) with a stable gap. (b) Concentration dependence of normalized maximum resistance change showing the saturation of 2-APB inhibition at 200 μM (50 μM (n = 5), 100 μM (n = 6), 200 μM (n = 8), and 400 μM (n = 9)). The error bars are the standard errors.
Simultaneous measurement of the electrical coupling and dye diffusion in response to application of 400 μM 2-APB. (a) Time course of changes in resistance (red triangle, left axis) and the fluorescence intensity (blue square and green dots, right axis), showing that both conductance and dye diffusion was inhibited by 2-APB. The drug (400 μM 2-APB) was applied to the central stream during the periods indicated by double headed arrows in (a). For diffusion, CFDA (5 μM) was loaded to the right side solution at the time indicated by green arrow in (a). (b) Fluorescent image showing the boundary (red dashed line) between the dye loading stream (right side) and the central stream (left side). The fluorescence intensity was measured from two windows (yellow outlined in b) centered at 25 and 75 μm from the boundary in the gap region. (c) Derivative of the fluorescence intensity at 25 μm with respect to time (red square, right axis), showing that 2-APB diminished the changes in dye intensity. The change in fluorescence intensity at 25 μm (blue squares) is also plotted for comparison. The data show higher sensitivity of the electrical measurement compared to the diffusion measurement.
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