Homogeneous penetration of EpCAM-FITC antibody in whole spheroids. (A) Flow cytometry analysis of CT320X6 (left histogram) and HT29 (right histogram) cells from dissociated spheroids. Cells were labelled with the same FITC-conjugated EpCAM antibody (red area) or FITC-conjugated isotype control antibody (white area). All cells were EpCAM positive. (B) CT320X6 (left panels) and HT29 (right panels) spheroids were fixed and permeabilized as described in Methods. Spheroids were then directly labelled using FITC-conjugated EpCAM antibody (yellow) and counterstained with TOPRO-3 (cyan). Acquisitions were taken at 40 μm and 70 μm depth within the same spheroid (scale bar = 50 μm).