2.7. Cellular Uptake Study Using Fluorescent Microscopy and Flow cytometer

We conjugated fluorescein-NHS (excitation/emission: 498/517 nm; Lumiprobe, Cockeysville, MD) to Man-PEI and complexed it with pGL-3 plasmid. We investigated the cellular uptake using (a) fluorescence microscopy with an Olympus BX63 microscope, and (b) flow cytometry using a BD FACS Calibur Flow Cytometer along with CellQuest Pro software. Briefly, for fluorescent microscopy, we incubated SW480 and HCT-15 cells with Man-PEI-fluorescein complexed with pGL-3 plasmid at N/P ratio 20:1 in chambered cell culture slides (Falcon, Corning, NY, USA). Following predetermined incubation periods, cells were washed with 1× PBS, fixed with 4% formaldehyde, and covered with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting media before visualizing under the microscope.

For the flow cytometric analysis, we incubated the SW480 and HCT-15 cells in the presence of Man-PEI-fluorescein/pGL-3 complexes, as described above, for predetermined periods. Subsequently, the cells were washed with 1× PBS, harvested, fixed with 4% formaldehyde and analyzed under a flow cytometer.