Fig. 2. Observation of Rex1-GFP/Oct3/4-CFP double knock-in ES cells by microscopy and flow cytometry. (A-H) Morphology and fluorescence of colonies of Oct3/4⁺/Rex1⁺ and Oct3/4⁺/Rex1^- cells in culture of Rex1-GFP/Oct3/4⁺-CFP double-knock-in ES cell line under puromycin selection (selection for cells expressing Oct3/4). (A,C,F) GFP fluorescence, (D,G) CFP fluorescence and (B,E,H) bright field. (A,B) Low magnification of knock-in ES cells. A small number of GFP (Rex1)-negative cells was observed. Arrows in B indicate colonies of Rex1^-/Oct3/4⁺ (GFP^-) cells. (C,D,E) High magnification of colonies in which Rex1⁺ cells were dominant. (F,G,H) High magnification of colonies in which Rex1^- cells were dominant. (I,J) Result of analysis of knock-in cells by flow cytometry. (I) Comparison of GFP fluorescence of Rex1-knock in cells with the parental cell line (OLC2-1). (J) GFP fluorescence of Rex1 knock-in cells disappeared within 3 days when differentiated by withdrawal of LIF. (K) Rex1 knock-in cells were stained with anti-PECAM-1 monoclonal antibody conjugated with PE and analyzed by flow cytometry. Most GFP-positive cells reacted with PECAM-1 antibodies. Scale bars: 50 µm.