Fig 2: Internalisation studies of novel mAbs KU44.22B and KU44.13A in BxPC-3 and AsPC-1 human pancreatic cancer cells determined by (A,B) Immunofluorescence, BxPC-3 and AsPC-1 cancer cells were grown to near confluency and incubated with purified mAbs KU44.22B and KU44.13A respectively (50 μg/ml) or control (PBS/1% BSA) at 4 °C for 1 h and subsequently at 37 °C for extra 30 min to allow internalisation. Cells were then fixed, permeabilised and incubated with anti-mouse secondary antibody (Alexa Fluor 488; 1:200) at 4 °C for 1 h for detection using Nikon eclipse i80 microscope; and (C,D) ELISA, BxPC-3 and AsPC-1 cancer cells were grown to near confluency 96-well plates and incubated with purified mAbs KU44.22B and KU44.13A respectively (50 μg/ml) or control (PBS/1% BSA) at 4 °C for 1 h and subsequently at 37 °C for extra 30 min to allow internalisation. Cells were then fixed, permeabilised and incubated with HRP-conjugated rabbit anti-mouse (1:1000, STAR13B, AbD Serotec) and the absorbance of each sample measured at 450 nm.