Fig. 3

Mouse and humanized antibodies mDC8E8/AX004 do not influence neuronal viability and physiological ATP production. a Representative image of human neuroblastoma cell line SHY5Y with internalized fluorescently labelled AX004 Alexa Fluor 546 antibody (60 μg) 24 h post electroporation. Microtubules were decorated with AX004 Alexa 546, thus confirming the specific intracellular binding site. b Fluorescent image of rodent primary neurons stained with Hoechst (c = 5 μg/mL, 30 min incubation live cells at 37 °C) 24 h post electroporation with unrelated control mAb DC51 or AX004 mAb. Scale bar, 20 μm. c Quantification of neuronal viability 24 h post electroporation with AX004 antibody. Non-pycnotic nuclei were counted as viable, as determined by Hoechst 33358 staining (1 μg/mL) and expressed as a percentage of total (n = 2 independent experiments in hexaplicate). Data are presented as mean ± SEM. No statistically significant change between groups stated as ns. d Primary neurons 24 h post electroporation (MaxCyte Technology) with AX004 (60 μg) were lysed and intracellular ATP levels were measured with CellTiter-Glo® Luminescent Cell Assay (Promega) based on manufacter’s instructions. The experiment was repeated from 2 independent preparations in triplicates. Data are represented as arbitrary units (AU) and shown as mean ± SEM. No significant change between groups stated as ns