Fig. 6. Colonies formed from E14.5 Dlk⁺ cells consist of albumin⁺ and CK19⁺ cells. (A-C) Dlk⁺ cells were isolated from E14.5 fetal liver by AutoMACS and cultured on chamber slides coated with type IV collagen at a density of 50 cells/cm² in the presence of HGF and EGF. After 5 days of culture, cells were fixed and stained with anti-albumin and anti-CK19 antibodies. Expression of albumin and CK19 was visualized with Cy-3 and FITC, respectively. Various sizes of colonies (A, a small colony containing about 40 cells; B, a medium colony containing about 60 cells; C, a large colony containing over 100 cells) were formed in a low density culture, which contained both albumin⁺ (red in A-1, B-1 and C-1) and CK19⁺(green in A-2, B-2 and C-2) cells. Both images were merged in A-3, B-3 and C-3. (D) A large colony formed from Dlk⁺ cells was picked from the culture dish and mounted on a glass slide by cytospin. The cells were stained with anti-albumin and anti-CK19 antibodies. Some of them expressed both albumin (red in D-1) and CK19 (green in D-2). In D-3, both images were merged. (E) A single Dlk⁺ cell was isolated and inoculated in one well of a 96-well plate coated with type IV collagen by FACSvantage. A large colony derived from a single Dlk⁺ cell consisted of albumin⁺ (red in E-1) and CK19⁺ (green in E-2) cells. Both images were merged in E-3. Bars, 100 µm.