Gfi1b:GFP expression during erythroid development in bone marrow and cultured megakaryocytes. (A) Living cells from bone marrow of Gfi1b^+/GFP mice were stained with CD71-PE and Ter119-APC and subjected to FACS analysis (left). Cells were gated for different developmental stages, and GFP expression of these cells is shown in a histogram (right). (B) Living cells from bone marrow of Gfi1b^+/GFP mice were stained with Ter119-APC and sorted for low, medium, or high GFP expression (left). Transcripts from the Gfi1b allele (▪) and the GFP allele (⊡) were quantatized by real-time PCR. Shown are the Δct values where the Gfi1b or GFP transcripts become detectable relative to the transcripts of the glyceraldehyde-3-phosphate dehydrogenase gene on the same threshold line. (C) Megakaryocytes were generated from bone marrow of Gfi1b^+/GFP mice by cultivation in DMEM in the presence of 10 ng/mL thrombopoietin (TPO) and 10 ng/mL IL3 for 5 days. Cytospins were prepared from nonadherent cells, and GFP expression was detected by fluorescence microscopy (right). Afterward, cells were stained according to the Wright-Giemsa protocol and photographed using visible light (left). (Rightmost) Flow cytometric analysis of c-Kit expression and GFP fluorescence in bone marrow–derived, large CD41-expressing cells. Cells were analyzed using a Leica DM IRB fluorescence microscope with an L 20×/0.4 NA Corr PH1 objective lens and photographed using a Nikon Coolpix 990 digital camera (Nikon, Düsseldorf, Germany) and LM-scope Adapter (Micro Tech Lab Rudnicki KEG, Graz, Austria). (D) Flow cytometric analysis of bone marrow cells after depletion of Lin⁺IL7Rα⁺Sca-1⁺ cells. Gatings for highly pure megakaryocyte progenitor population and expression of green fluorescense among these cells are shown. The data shown is representative of 3 similar data obtained from individual experiments.