Scrape Loading and Dye Transfer Assay for Gap Junction Intercellular Transference

Scrape loading and dye transfer assay was performed to detect the gap junction intercellular transference in the living cells as previously described.^{21 (link),23 (link),44 (link)} Briefly, small molecular (MW < 900) dyes (Lucifer yellow, MW457, L0259, Sigma) were introduced and their intercellular movement through gap junctions was detected by CLSM. Firstly, the adherent cells were treated with TGF-β1 as experimental group. After treatment, cells are required to grow to a full confluence (100%), and then were washed with CaMg-PBS and then three scrapes were made using a surgical blade needle in the presence of 1 mg·mL⁻¹ Lucifer yellow (L0259, Sigma). Cells were incubated for 7 min at room temperature. After rinsing thoroughly to eliminate background, dye transfer was captured using CLSM.