Phrodo red avidin

αCD33-AM_Fab was labeled with pHrodo Red Avidin (ThermoFisher Scientific, Waltham, MA) according to manufacturer’s instructions. MV4-11 cells were labeled with 500 ng/ml αCD33-AM_Fab-pHrodo and 1:1000 Hoechst 33342 (ThermoFisher Scientific) for 15 min at 4 °C. Unbound AM was removed and cells were incubated for 6 h at 4 °C or 37 °C on poly-D-lysine-coated glass-bottomed two-well ibidi slides (ibidi GmbH, Gräfelfing, Germany). Images were acquired on a Nikon TiE microscope. Instrument settings are outlined in detail in the supplements.

Biotinylated ch2448 were incubated with equimolar of pHRodo Red Avidin (Thermo Fisher Scientific, #P35362) in the dark and on ice for 5 min. The conjugated mAb (20 μg) was added to the hESC culture and real‐time visualization of the internalization was carried out by video capture on a DeltaVision (GE Healthcare Life Sciences). For the counter‐staining with Phallodin, after the internalization of ch2448 conjugated to pHRodo, the hESC culture was washed twice with cold PBS and subsequently fixed with 4% paraformaldehyde/PBS for 15 min. The cells were washed twice with cold PBS and permeabilized with 0.5% Triton‐X/PBS for 10 min. The washing was repeated and cells were blocked with 10% FBS/PBS for 10 min. The cells were washed twice with PBS and incubated with Phallodin conjugated to AlexaFluor 488 for 30 min in the dark. Excess dyes were washed off with PBS and 500 µl 1% BSA/PBS was added to each well before imaging.

Sub-confluent cells were harvested and plated in 50 μl per well at the following concentrations: MIA PaCa-2 (8 x 10³ cells/well), A-172 (8 x 10³ cells/well) and SKOV3 (1 x 10⁴ cells/well). Cells were then incubated overnight at 37°C, in 5% CO₂. On day 1, coupling of the antibodies was performed by incubation of biotinylated PODO447 or palivizumab control mAb (40 (link)) with pHrodo™ Red Avidin (ThermoFisher, #P35362) at 1:1 molar ratio for 30 min at 4°C. Next, 50 μl of cold pHrodo™-mAb mix was added to the cells and the plate was placed in the Incucyte®. The Incucyte® ZOOM software was set in “standard” mode with the phase and red channels selected and set to scan every 10 min for 24 hours. The rate of antibody internalization was evaluated through increased fluorescent area, reported as total internalization area (μm²/well). Average internalization was calculated, and statistical analyses was performed using two-way ANOVA in GraphPad Prism software.

Antibody internalization was measured using OVCAR-3 and OV-KD cells and the PHrodo™ Red Avidin diagnostic method following the manufacturer’s instructions (ThermoFisher). PHrodo Red-labelled antibodies were generated by adding 25 μg/mL of PHrodo Red Avidin to 25 μg/mL biotinylated or non-biotinylated (negative control) antibodies for 30 minutes at room temperature in microfuge tubes. Upon completion, reactions were centrifuged at 10,000 g for 2 minutes to remove aggregates. Target cells were seeded in triplicate at 10,000 cell/well in 96-well opaque plates and grown for 48 hours in R7.5 media at 37°C in 5% CO₂. To monitor internalization, PHrodo Red-labelled antibodies and controls were added at a final concentration of 5 μg/mL to 4°C chilled R7.5 media. Next, PHrodo Red-labelled antibodies were added to appropriate wells and plates were incubated on ice for 1 hour to minimize immediate cellular uptake. Wells were then washed twice with 4°C DPBS to remove unbound PHrodo-Red or control antibody. Plates were incubated at 37°C in 5% CO₂ and monitored for uptake via fluorescence over a 24-hour period using a Varioskan plate reader.

Apoptotic MLE cells were labeled with Annexin V-Biotin (Miltenyi Biotech inc., CA),and then incubated with pHrodo Red Avidin (Invitrogen). The pHrodo stained apoptotic cells were administered intranasally (10⁶ cells/mouse). BAL cells were collected and stained with DAPI and CD11c and subjected to confocal microscopy. Zeiss confocal system (LSM 510 META) with an inverted microscope was used for imaging. The images were processed using LSM Zen 2009 software and compiled using Adobe photoshop software.