Figure 1: Generation of monoclonal antibody against the CTOS surface.

(a) Top, whole mount immunocytochemistry (ICC) without permeabilization of live CTOSs with 5G2 mAb. CTOSs were incubated with 1 μg/ml purified 5G2 mAb or mouse IgG3κ isotype control and observed by confocal microscopy. Scale bar, 50 μm. Bottom, phase contrast images of an adhesion assay. CTOSs were cultured for 5 days with or without 5G2 hybridoma culture media on a type I collagen-coated dish. Scale bar, 100 μm. (b) Quantification of adhesion to type I collagen-coated plates based on the percentage of adhered CTOSs. Data indicate mean ± SD from three independent experiments. Each condition was performed in triplicate and data are shown relative to the negative control (non-treated) in each experiment. Vehicle: 10% glycerol-PBS. Differences between 5G2 and control IgG were analysed using ANOVA. (c) Immunohistochemistry of sectioned CTOSs with 5G2 mAb (green) and ZO-1 (red). CTOSs were cultured in floating or gel embedded conditions. Gel embedded CTOSs were cultured for 3 days in type I collagen gel. Scale bar, 50 μm. (d,e) Effect of pre-incubation with 5G2 mAb on CTOS growth under Matrigel embedded (d) or floating (e) culture conditions. Growth of CTOSs was evaluated by the day 7 to day 0 size ratio and the data normalized to a mean of 0 μg/ml for each experiment. Values are relative to the negative control (non-treated). Statistical analysis was performed by the Kruskal-Wallis test. Horizontal bar: median; boxes: 25^th and 75^th percentiles; bars: 10^th and 90^th percentiles. Data are from more than three independent experiments. V, vehicle, 10% glycerol-PBS.