Binding and internalization of Cal-CD19-liposomes. (A) Binding and internalization by SUP-B15 cells. Cells were incubated with Cal-CD19-liposomes at 37° C for 10 (left) or 60 (right) minutes and were analyzed by FCM to detect calcein fluorescence. In addition, cells were treated with (thin solid line) or without (bold line) pronase to remove surface-bound liposomes. When those liposomes were internalized into the cells, calcein fluorescence was expected to remain. Untreated cells were used as a negative control (broken line). (B) Internalization efficiency of liposomes by Ph⁺ ALL cell lines. These cell lines were incubated with Cal-bare-, Cal-PEG-, or Cal-CD19-liposomes at 37°C for 60 minutes and treated with pronase. The rate of calcein-positive cells was determined by FCM. (C) Blocking of Cal-CD19-liposome internalization into SUP-B15 cells by CLB-CD19 antibody. SUP-B15 cells were incubated with 10 (left), 100 (middle), or 1000 (right) μg/mL unlabeled CLB-CD19 antibodies at 37°C before incubation of the Cal-CD19-liposomes. Thereafter, cells were treated with pronase and assessed by FCM (thin solid line). Untreated cells (broken line) and cells not treated with CLB-CD19 antibody (bold line) were used as negative and positive controls, respectively.