Flow cytometry analysis and confocal microscopy observation of MIL5scFv-Arg9 to penetrate NIH3T3 cells (HER2 negative). Cells were incubated with diluted MIL5scFv-Arg9-FITC or MIL5scFv-FITC at 37°C for 2 h and then detected by flow cytometry. A: Cells treated with herceptin-FITC as negative control; B: Cells treated with MIL5scFv-Arg9-FITC; C: Cells treated with MIL5scFv-FITC. The intensity of fluorescence signal was evaluated with MFI value marked on the right top of each panel. According to the fluorescence intensity, Arg9 could help MIL5scFv penetrate NIH3T3 cells. Data are representative of two independent experiments. D: Confocal microscopy observation of MIL5scFv-Arg9 to penetrate NIH3T3 cells. Cells were incubated with PBS (Control) or herceptin-FITC as negative control, MIL5scFv-Arg9-FITC (1:10) or MIL5scFv-FITC (1:10) at 37°C and collected consecutively in 0.5 h, 1 h, 2 h and 5 h. The distribution of fluorescence signal in the cells was observed under a laser scanning confocal microscopy. Fluorescent signal could be detected after 0.5 hour in MIL5scFv-Arg9 treated samples, while in MIL5scFv treated samples, weak signal could be seen after 5 hours. Each panel was merged with two photos: FITC conjugated protein (green) and nuclei (blue, cells were stained with Hoechst 33258), which were not shown respectively here.