To analyze the affinity and internalization of the anti‐TF mAbs and ADCs, the mAbs and ADCs were directly conjugated with Alexa647 using a monoclonal antibody labeling kit (Invitrogen) according to manufacturer's instruction. The affinities of the mAbs and the ADCs against pancreatic cancer cells were analyzed by flow cytometry. Inside a 2 ml tubes, 2 × 105 harvested cells were incubated with 0.2 μg of each Alexa647‐conjugated mAbs and ADCs for 30 min at 4°C. After washing with PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA (B. E. PBS), the cells were nuclear stained with a propidium iodide (PI) solution (Invitrogen, Eugene, OR). The stained cells were analyzed by flow cytometry using Guava easyCyte (Millipore, Billerica, MA).
To analyze the internalization of the anti‐TF ADC into cancer cells, 3 × 103 cells of BxPC‐3 were pre‐cultured in flat‐bottomed 96‐well tissue culture plates (Corning, Corning, NY) overnight. Then, 0.2 μg of Alexa647‐conjugated ADC were added to the cells and incubated at 37°C for 0 and 3 hr. To identify the lysosomes, the cells were also incubated for 1 hr with 50 nM of LysoTracher (Invitrogen). After rinsing with PBS, the cells were fixed with 4% paraformaldehyde (Wako) for 10 min and then nuclear stained with a 4′6‐diamidino‐2‐phenylindole‐2HCl (DAPI) solution (Roche, Basel, Switzerland).
To analyze the internalization of the anti‐TF ADC into cancer cells, 3 × 103 cells of BxPC‐3 were pre‐cultured in flat‐bottomed 96‐well tissue culture plates (Corning, Corning, NY) overnight. Then, 0.2 μg of Alexa647‐conjugated ADC were added to the cells and incubated at 37°C for 0 and 3 hr. To identify the lysosomes, the cells were also incubated for 1 hr with 50 nM of LysoTracher (Invitrogen). After rinsing with PBS, the cells were fixed with 4% paraformaldehyde (Wako) for 10 min and then nuclear stained with a 4′6‐diamidino‐2‐phenylindole‐2HCl (DAPI) solution (Roche, Basel, Switzerland).