Internalization of PhD targeted-phage is dose dependant in CP cells in vitro. TR-CSFB cells were incubated with increasing concentrations of Peptide D-targeted phage for 2 h, at 37C. Cells were acid washed to remove cell surface binding, immunostained with antibodies against M13 phage and subsequently detected using Alexa594-labeled goat anti-rabbit antibodies. At 1 × 109 pfu/ml, no significant intracellular staining was observed (Panel A). At 1 × 1010 pfu/ml (Panel B), there was detectable immunoreactivity for M13 in the cytoplasm which clearly increased when the cells were incubated with 1 × 1011 pfu/ml (Panel C). This experiment shows that there was a correlation between phage titer and the intensity of intracellular immunoreactivity indicating that internalization is dose dependant. Internalization of targeted-phage was also time-dependant (Panels D-E). TR-CSFB cells were incubated with Peptide D-targeted phage (5 × 1010pfu/ml) at 37C, acid washed, immunostained with M13 antibodies and internalization visualized using Alexa594 labeled secondary antibodies. Cells were counterstained with DAPI to visualize the cell nuclei. By 15 min, Peptide D-targeted phage were already internalized (Panel D) and this intracellular staining increases over the following 2 h (Panel E) but particle is degraded by 48 h (Panel F). M13 phage: Red; Cell nuclei: white.
Gonzalez et al. BMC Neuroscience 2011 12:4 doi:10.1186/1471-2202-12-4