Internalization of Sm and PtC by primary leukemic cells. Eμ-TCL1/IgSm leukemia cells were stained at 4°C with biotin-labeled Sm, washed, and then stained with streptavidin-FITC. Eμ-TCL1/IgPtC leukemia cells were stained at 4°C with fluorescein-labeled PtC-liposomes. Cells were then incubated for 20 minutes at 4°C or 37°C, fixed, and costained with cholera toxin B (CTB)-Alexa 633 to identify the cellular membranes and Hoechst 33332 to mark the nuclei. Images were acquired with a laser scanning confocal microscope (TCS SP5; Leica Microsystems, Mannheim, Germany) using a ×40 (NA = 1.25) oil immersion lens with optical pinhole at 1 AU. Confocal Z-stacks collected at a magnification of ×40 with a 0.29-μm interval and a 5-μm total optical depth are displayed. Scale bar indicates 5 μm. Images at 4°C show Sm/CTB and PtC/CTB colocalization on the cellular membrane. In images acquired from cells incubated at 37°C, Sm and PtC can be seen dissociated from CTB and located below the cellular membrane. IgHEL-expressing normal B cells incubated either with the Sm antigen or PtC were used as a negative control in these experiments (data not shown).