Apc anti mouse cd326

All flow cytometry was performed on a Beckman Coulter Gallios 6C.
Cell Culture RAW264.7 cells (ATCC) were grown to $70% confluence in Dulbecco's Modified Eagle Media (DMEM) (Sigma) containing 10% FCS and passaged using a cell scraper. Mouse tumor cell lines (4T1 and CT26) were grown in monolayers in RPMI-1640 media (Sigma) containing 10% FCS and passaged 2-3 times per week using phosphate buffered saline (PBS)/Trypsin EDTA. All cells were maintained in culture at 37 C in a humidified atmosphere containing 5% CO 2 and routinely screened and found to be free of mycoplasma. EpCAM expression and receptor number in the mouse cell lines was quantified using flow cytometry with APC anti-mouse CD326 (Biolegend) using Quantum Simply Cellular anti-Rat IgG microspheres (Bangs Laboratory). As CT26 were shown to be negative for EpCAM, cells were transfected with a pcDNA3.1+C DYK vector containing the mouse EpCAM ORF clone (NM_008532.2) (Genescript) using Lipofectamine 2000 (Thermo Fisher). G418 selection was used to obtain pure populations of EpCAM expressing CT26 clones, and cells were screened as described above for EpCAM expression. Clones were examined for growth rate, drug sensitivity and in vivo tumorgenicity, and one that possessed high EpCAM expression with the above 3 parameters being similar to the parental CT26 cell line was selected for all subsequent studies (CT26Ep12.1).