Fig. 3. Formation of feeder-free colonies is dependent on NANOG expression. (A) Five-hundred cells per cm² from H9 and H13 cell lines were seeded in 12-well plates coated with gelatin and FCS. After 8 days, the cells were fixed and stained for alkaline phosphatase (AP) activity. Positively stained colonies were counted. The results shown for H13 clones represent the average of three independent clones. Scale bars represent s.e.m. (^*P<0.05). (B) Representative photos of each clone. AP staining of undifferentiated cells appears as red staining. (C) The experiment described in A was repeated after establishment of revertant clones from which the transgene was removed using the CRE-loxP system. Shown are results of the parental cells and the average of three individual CRE clones derived from it. Error bars represent s.e.m. (^*P<0.05). (D) Representative photos of the results depicted in C.