Fig. 4. Flow cytometric analysis of Dlk expression and isolation of Dlk⁺ cells. (A) E14.5 liver cells were stained with anti-Dlk mAb and analyzed by FACS. Dlk⁺ cells were separated from Dlk^- cells, which mainly consisted of hematopoietic and endothelial cells. Dlk⁺ cells were about 10% of total E14.5 hepatic cells. (B) Dlk⁺ cells isolated by AutoMACS were stained with anti-AFP (1) and anti-albumin antibodies (2). The morphologies of Dlk⁺ cells are shown in the upper panels. Over 95% of Dlk⁺ cells were stained with anti-AFP and anti-albumin antibodies (red in lower panel). (C) cDNA was synthesized from total RNA of CD45^-TER119^-Dlk^- and CD45^-TER119^-Dlk⁺ cells separated by FACSvantage. Quantitative PCR reaction was performed with a LightCycler. The signals for AFP and albumin were normalized with the GAPDH level and relative expression level (the expression level in Dlk^- cells = 1) are shown. The mRNA levels for AFP and albumin in Dlk⁺ cells (white bar) were 60 and 40 times, respectively, those in Dlk^- cells (black bar).