Figure 4: Recognition of CT26 cells by sera from HCF-immunized animals. (a) Histogram plots showing antibody recognition for membrane and cytosolic antigens on CT26 cells. Flow cytometry analyses were carried out on permeabilized or nonpermeablized CT26 tumor cells incubated with sera (diluted 1 : 100) collected from animals immunized with human HCF. Controls consisted of preimmune sera (PI) or cells incubated with secondary antibody only (Ctl). Five thousand events were collected and gated on FSC versus SSC dot plot. (b) Median fluorescence intensity (MFI) representing antibody recognition of membrane and cytosolic antigens on nonpermeabilized and permeabilized CT26 cells, respectively. In this case, different sera dilutions (1 : 50, 1 : 150, and 1 : 450) were used, and MFI values were subtracted to the corresponding preimmune sera at the same dilution.