Subcellular localization of CD44-GFP and CD133-GFP fusion proteins in transfected CD34⁺ cells. Using the new Amaxa nucleofection technology, human CD34⁺ cells enriched from umbilical cord blood were transfected with the expression plasmid encoding either for YFP (Ci), CD44-GFP (Di-ii) or CD133-GFP (Ei-ii) under the control of the cytomegalovirus promoter. Transfected cells and, as negative control, untransfected cells (B) were cultivated for 1 day in serum-containing medium supplemented with early acting cytokines and were analyzed by flow cytometry (A-B,Cii,Dii,Eii) and fluorescence microscopy (Ci,Di,Ei). (A-B,Cii,Dii,Eii) A representative experiment (n = 5) of the CB-derived cells untransfected (B) or transfected with different expression plasmids (Cii,Dii,Eii) was stained with a PE-conjugated anti-CD34 antibody and analyzed by flow cytometry. The cells analyzed in panels B, Cii, Dii, and Eii were gated according to the morphology depicted on a forward scatter/side scatter plot (A). Note, cells shown in panel Cii are extremely positive for YFP; most of them stick to the right border of the plot. (Ci,Di,Ei). Differential interference contrast image shows fluorescence overlay of YFP (Ci), CD44-GFP (Di), or CD133-GFP (Ei) in living transfected CD34⁺-enriched cells. The YFP is strongly expressed throughout the cytoplasm of the cells, whereas CD44-GFP and CD133-GFP are concentrated in the uropod of the migrating cells (arrows in Di and Ei). The white arrows indicate the cells shown in the insets (high magnification).