Flow cytometric analysis for expression of the FR. (Top) CD34⁺ cells and KB cells were incubated with rabbit preimmune serum or rabbit polyclonal antibody to placental FR for 30 minutes, washed, and incubated with secondary antibody (goat anti-rabbit IgG-FITC) for 30 minutes to evaluate FR expression on each cell population. Cells were then analyzed by fluorescence flow cytometry, where log cell fluorescence is plotted on the x-axis and cell number on the y-axis. The filled peak corresponds to cells treated only with preimmune serum, while the open peak shows cells labeled with anti-FR IgG. The results indicate that both CD34⁺ cells and KB cells express FR on their cell surfaces. (Bottom) CD34⁺ cells and KB cells were incubated with FA-PEG-liposomes or PEG-liposomes containing calcein for 4 hours, washed, and analyzed by fluorescence flow cytometry. The filled peak represents PEG liposome staining and the open peak represents FA-PEG-liposome staining. For CD34⁺ cells, the peaks for FA-PEG-liposomes and PEG-liposomes overlay one another, showing no detectable binding to FR. In contrast, FA-PEG-liposomes bind well to KB cells.