A. FACS analysis of PBL transduced with various human or murine monoclonal antibody (mAb) based CARs. Five days following the transduction of PBLs with retroviral vectors expressing CARs targeting ERBB2, EGFRvIII, VEGFR2, CSPG4 antigen or CD19, cells were first stained with biotinylated protein L followed by PE-conjugated streptavidin (SA). Untransduced (UT) cells and T cell receptor (TCR) transduced cells were used as negative controls. Cells were analyzed using a FACScan flow cytometer and the data analyzed with FlowJo software. B. Comparison of Protein L staining to other established methods of detection of CAR expression. PBL expressing murine mAb-based anti-CSPG4 CAR were analyzed for CAR expression using goat-anti-mouse fragment antigen binding (Fab) versus protein L. These results are representative of five independent determinations. C. Humanized mAb-based anti-ERBB2 CAR transduced PBL were analyzed for CAR expression using a CAR-specific ERBB2-fragment crystallizable (Fc) fusion protein versus protein L. The result presented here is a representative experiment of three independent determinations.