Scrape loading

For scrape loading of confluent monolayers of differentiated P19 embryonic carcinoma cells (Doble et al., 1996), cells were washed three times with calcium and magnesium free PBS, and covered with 1mg/ml Lucifer Yellow - CH (Sigma-Aldrich, Steinheim, Germany) and 1 mg/ml Rhodamine - dextran (Sigma-Aldrich, Steinheim, Germany) in PBS at 37°C. Four parallel scrapes were set in the monolayer with a scalpel blade, with the dye solution being removed after 2 min, and the cells being washed three times with PBS. Four minutes after setting the scrapes, cells were fixed for 10 min with an ice-cold solution of 1% PFA in PBS to stop further spreading of the dye. After three washes with PBS, dye spreading was documented photographically under an inverse microscope, equipped with epifluorescence (Zeiss, Jena, Germany). For statistical evaluation distances of dye spreading was measured at 6 locations at each of the four scrapes for 6 independent experiments. Significance was analyzed by Student's two-tailed t-test.

Doble BW, Chen Y, Bosc DG, Litchfield DW, and Kardami E. 1996. Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes. Circ Res 79:647-658.