Confocal microscopy study of ITIM-dependent internalization of antibody-bound CD33 in transduced cell lines. OCI-AML3 cell sublines transduced with either wild-type CD33 or CD33 mutants were incubated with Alexa Fluor 594–labeled hP67.6 on ice water before the cells were transferred to antibody-free medium and either kept in ice water (left panels) or incubated in 37°C (right panels) to allow internalization of antibody-bound CD33 for 30 minutes. Cells were then fixed, stained with DAPI, and visualized by confocal microscopy.