Figure 2

EPISPOT assays and immunocytochemistry experiments. (a) Dual-fluorescent CK19-EPISPOT assay with two different couples of mAbs. Red and green immunospots represent protein fingerprints of cells releasing CK19 detected with anti-CK19 AE1^Alexa555 and Ks19.2^Alexa488 mAbs, respectively. Yellow fluorescent immunospots (Merge) are the results of dual staining with both mAbs. (b) Single-fluorescent CK19 Ks19.2^(Alexa488)-EPISPOT assay on MCF-7 cells without and with the addition of cycloheximide (50 μg/ml). (c) Dual-fluorescent CK19 Ks19.2^(Alexa488)/MUC1^(Alexa555) EPISPOT assay on the MCF-7 cancer cell line. Green and red immunospots represent CK19-RCs and MUC1-RCs, respectively. Yellow fluorescent immunospots (Merge) are the results of dual staining with both mAbs. (d) Immunostaining of the nucleus^(dapi), the MUC1^(Alexa555), and the CK19 Ks19.2^(Alexa488) of breast MCF-7 cells (left). Immunostaining of the nucleus^(dapi), the CK19 Ks19.2^(Alexa555), and the cleaved cytokeratin 18 with the M30 CytoDEATH^(FITC)of MCF-7 cells after an incubation with vincristine (20 μmol/l), an inducer of apoptosis (right). The antibody M30 CytoDEATH which recognizes a neo-epitope formed after caspase cleavage of cytokeratin 18 at Asp396 (CK18Asp396-NE M30 neo-epitope) during and after apoptosis, does not bind native CK18 of normal cells and is a very reliable and convenient tool for demonstration of apoptosis in single cells [41]. CK19 vesicles were detectable in the viable but not in the apoptotic MCF-7 cells.