Cell migration determined by cell scratch assay

A marker was used to draw three evenly spaced straight lines behind the 6-well plate as positioning lines. RF/6A cells were inoculated in 6-well plates in at 5x10⁵ cells per well. After 24 h of culture in vitro in a dish, a monolayer had formed and a 100-µl pipette tip was used to scratch the cells, perpendicular to the positioning line. The intersection point of the scratch and the positioning line were taken as the monitoring point. PBS was used to gently rinse the bottom of the culture well three times to remove any detached cells. Medium containing 10% fetal bovine serum (13,14) and different concentrations of visfatin and D-glucose were added according to the different group assignments. Under an inverted microscope, an image of the scratch width at the monitoring point was captured, which was considered as the 0-h time point. The cell culture was continued for 24 and 48 h and the width of the scratches at the monitoring points was recorded. ImageJ software version 1.7.0 [National Institutes of Health (NIH)] was used to calculate the width of the scratches. A total of five fields of view were selected for observation in each group. The independent experiments were repeated three times.