Colony assay of GFP⁺ cells in the bone marrow. (A) The Lin-/c-Kit⁺ fraction is subdivided into 4 fractions with the expression of GFP and CD71. The percentage of cells in each quadrangle is shown. Cells in each fraction were sorted and cultured with only Epo (B), Epo and SCF (C), or Epo, SCF IL-6, and IL-3 (D). Colonies were counted 3 days (B) or 7 days (C, D) after culturing. (E) A colony derived from CFU-E in the CD71⁺/GFP⁺ fraction was stained by benzidine (left panel, blue staining), and some of the cells in the colony were detected by GFP expression under the fluorescent microscope (right panel). (F) A mixed colony derived from a CD71^-/GFP⁺ cell was observed by bright (top panel) and fluorescent (middle panel) microscopy. GFP expression was detected in red cells (erythrocytes [E]) and large cells (megakaryocytes [Mk]) but not in small white cells (granulocytes [G]) and large extended cells (macrophages [Mc]). The top and middle panels were merged in the bottom panel (F). Original magnifications, × 200 (E) and × 100 (F).