Igo Primo For Windows Ce 6.0l

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Igo Primo For Windows Ce 6.0l

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Gap-junction permeability {#s4-9}
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For measuring the gap-junctional permeability, HUVECs were transfected with 6 µg of VE-cadherin-GFP. To label the plasma membrane, HUVECs were exposed to Hilyte Fluo-4, AM dye (Invitrogen, Carlsbad, CA) for 4 hr. The cells were then permeabilized with 20 µM amphotericin B in recording medium (Invitrogen) for 1 hr at 37°C. Cells were washed with recording medium, stained with calcein AM for 1 hr at 37°C and then imaged. Changes in fluorescence during application of ATP, ADP or UTP were quantified as described ([@bib21]).

Cell-free gap-junction assay {#s4-10}
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A cell-free gap-junction assay was used as described ([@bib31]). Human erythrocytes with GFP-labeled spectrin (hemoglobin free) (University of Göttingen) or tectalangeal-GFP (TetTox-GFP) ([@bib5]) (kind gift from Prof. A. Cidelli, University of Washington) were diluted to 1% in buffer containing 0.1% BSA in PBS. 10 μL of erythrocyte suspension and 1 μL of GUVs or VE-cadherin-GFP expressing erythrocytes were mixed and added to a heptacellular dish. Osmolarity was then adjusted with sucrose to 290 mOsm for GFP and 320 mOsm for
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