EphB4 gene expression indicated by β-galactosidase activity within EBs. (A) Two clones of each EphB4^+/– (EphB4^+/–1 and EphB4^+/–2) and EphB4^–/– ES cells (EphB4^–/–1 and EphB4^–/–2) were induced for EB differentiation for 4 days and analyzed for gene expression by RT-PCR. (B) Kinetics of β-gal expression in EphB4^+/– EBs. EphB4^+/– ES cells were allowed to differentiate into EBs in suspension cultures in Petri dishes. EB cells at day 2, 4, 7, and 9 were harvested, stained with FDG for β-galactosidase activity, and analyzed by flow cytometry. Dead cells were gated out by a DNA dye, DAPI, staining (data not shown). (C) Flow cytometric analysis of β-gal expression (β-gal⁺ cells) in day-4 EBs from control (wild-type), EphB4^+/– and EphB4^–/– ES cells. (D) X-gal staining β-galactosidase activity in day EBs was analyzed by staining with 5-bromo-4-chloro-3-indolyl b-galactoside (X-gal). Original magnification, × 4.