Figure 6
Efficient expansion of keratinocytes carrying EGFP with a combination of polybrene and Y-27632. (A) Determination of colony-forming efficiency of keratinocytes treated with various concentrations of Y-27632 for four days, after the first day of cultivation. A total of 2.5 × 103 keratinocytes were seeded and cultured for 12 days until the cultures were fixed and stained with rhodamine B. Bar, 10 mm. (B-D) Flow cytometric analysis of keratinocytes cultured on a feeder layer of 3 T3 fibroblasts with various concentrations of Y-27632. Keratinocytes (5 × 105 cells) were cultivated with various concentrations of Y-27632 during lentivirus transduction at multiplicity of infections (MOI) 1, and then the keratinocyte cultures were analyzed after seven days of transduction. ITGA6-positive and -negative cells were recognized as keratinocytes and 3 T3 fibroblasts, respectively. The quantitative representation of the effects of Y-27632 on keratinocyte proliferation (B), transduction efficiency into the keratinocytes (C) and the expansion of enhanced green fluorescent protein (EGFP)-expressing keratinocytes (D). The values (means ± SD) were determined based on results from triplicate experiments.