Scrape loading/Lucifer yellow transfer assay of gap junction channel activity

Cultured cells that had been treated as indicated above were first incubated at 20–22 °C for 10 min in HEPES buffered-saline 1 (HBS1: NaCl 140 mM, KCl 5.5 mM, CaCl₂ 1.8 mM, MgCl₂ 1 mM, d-glucose 10 mM, HEPES 10 mM, pH 7.35), then washed in Ca²⁺-free HEPES buffered-saline for 1 min. Cells were subsequently exposed to Lucifer yellow (LY, 1 mg/ml) for 1 min ¹¹, washed, and LY was allowed to diffuse through GJCs during 8 min. Six successive fluorescent images taken at the center of the scrape line were captured using an inverted epifluorescence microscope (Diaphot-Nikon, Tokyo, Japan) for this 8 min diffusion period. Area of fluorescence was quantified with Image J program (NIH software).