For internalization, induced Edox and Cdox cells at a density of 1 × 106 cells/mL were incubated with a 50 nM solution of antibodies coupled with NHS-Alexa Fluor 488 in a complete RPMI medium at 37 °C overnight. Cells were harvested with centrifugation at 300× g for 5 min at 4 °C, washed twice with 1 mL PBS at RT, and stained with Hoechst 33,342 (BIO-RAD), diluted to 1 µg/mL in PBS for 5 min at RT. Staining was completed by rinsing cells twice with 1 mL of ice-cold PBS. For the measurement, cells were resuspended in 400 µL ice-cold PBS, and 10 µL of cell suspension were delivered into a slot of a 4-well microinsert in 35 µm µ-dish (Ibidi, Gräfelfing, Germany). Samples were analyzed with a DMI6000B microscope (Leica, Wetzlar, Germany) using HCX Objective Plan-Apochromat 63×/1.4 Oil. Data on sample fluorescence were processed using Leica Application Suite X software, version 3.7.0.
The control experiment to monitor cell surface staining proceeded in a similar way, except for antibody incubation, which was for 30 min on ice.
The control experiment to monitor cell surface staining proceeded in a similar way, except for antibody incubation, which was for 30 min on ice.