Application of FMv2CD44v8 for high throughput screening of exon skippable ASO. (A) To examine the applicability of FMv2CD44v8 for high throughput screening, a 96 well plate was separated into three parts and seeded with HeLa cells. After transfection of FMv2CD44v8, seven different ASOs were randomly and blindly added to the culture medium. Fluorescence was analyzed in each well and the exon skipping index was calculated after the construction of histograms. The index of each well is shown by a color; wells with less than 10, 10–30, 30–50, 50–90, and more than 90 of the index are illustrated by open, plaid green, vertical stripe green, dotted green and dark green circles, respectively. In the center of the circles, the corresponding ASO is indicated. NT denotes non-transfection of minigenes. In three separated regions, the wells with identical treatment disclosed similar index values. This indicated that multiple samples can be analyzed simultaneously. (B) The exon skipping index was summarized to reveal ASO-specific activity. The mean values are shown by vertical columns with error bars. Significant differences were detected by comparing the wells with the indicated ASOs and wells without ASO (ASO-) (* p < 0.05 and ** p < 0.001).