Scrape loading–dye transfer assay

Lucifer Yellow in Hank’s Buffered Salt Solution (HBSS) (1 mg/ml) or Rhodamine 123 in H₂O (1 mg/ml) was diluted 1:100 in FB-Plus and added to MA104 or jHIE cell monolayers. Monolayers were scraped with a 26 g needle and incubated for 5 min at room temperature. Monolayers were then washed three times with FB-Plus and imaged. The mechanical disruption causes cells adjacent to the scrape to take up dye, which then is permeable to adjacent cells if there are functional gap junction channels. Images are representative of each experiment performed in triplicate.