2.12. Endocytosis Inhibition Assay

50,000 cells per well were seeded on 24-well plates in serum-containing DMEM 24 h prior to treating them for 30 min with the following inhibitors: 4 µM cytochalasin D for macropinocytosis inhibition; 15 µM chlorpromazine for clathrin-mediated endocytosis inhibition; 12.5 µM nystatin for caveolae-mediated endocytosis inhibition; 6 mM 2-Deoxy-D-Glucose and 10 mM NaN₃ for ATP depletion (-ATP). Thereafter, cells were treated with hPep3/AF568-SSO complexes at 200 nM final concentration for 4 h in the presence of the inhibitors. Cells were washed twice with cold DPBS containing 100 µg/mL of heparin and detached with 0.05% trypsin-EDTA. The cell pellet was further washed with cold DPBS, and thereafter lysed in 0.1% Triton X-100. The endocytosis inhibition was evaluated by fluorescence spectroscopy and evaluated over hPep3/AF568-SSO treatments without inhibitors.