Analysis of Tumor Cell-Specific IgM Antibodies in Sera of CT26 Colon Tumor-Bearing Mice

For determination of tumor cell-specific IgM antibodies, indirect immunofluorescence analysis was used. Mice sera were thawed on ice and 1 μl of the respective serum sample was co-incubated with 1 × 10⁵ viable CT26 cells for 1 h at 4°C. Thereafter, cells were washed with PBS/10% FBS. The amount of bound antibodies was analyzed by adding staining solution [5.8 μg/ml FITC-conjugated goat anti-mouse IgM (Invitrogen, Darmstadt, Germany)] for 1 h at 4°C in the dark. After washing, cells were resuspended in PBS/10% FBS. Using flow cytometry, the mean fluorescence intensity of CT26 cells per sample was analyzed and equated with the tumor cell-specific IgM antibody level in the serum.