Detection of trogocytosis by confocal microscopy and flow cytometry. (A) Biotinylated U266 plasma cells stained with streptavidin Alexa Fluor 594. Images were acquired on a Zeiss LSM 510 Meta Confocal microscope (Carl Zeiss). (B) Anti-CD3 Alexa Fluor 488 stained T cells. (C-E) Coculture demonstrating the variable (low, intermediate, and high) density of biotinylated U266 membrane proteins acquired by CD3⁺ cells. (F) Biotin-strepavidin PE transfer to CD3⁺ (35%) and (G) CD3⁻ cells (2.0%). (H) Biotin transfer from U266 plasma cells to normal PBMC (n = 4) and myeloma PBMC (n = 6) was similar with greater transfer to T cells than to B or NK cells. Coculture with the other chronic B cell malignancies (CLL and Waldenstrom macroglobulinemia) resulted in less than 1% acquisition by T cells (n = 5). (I) Representative flow scatterplots of the biotin/strepavidin staining of U266 plasma cells and expression after coculture showing greater transfer of biotinylated proteins from U266 cells (bottom left panel) to CD3⁺ cells than CD19⁺ and CD16⁺ cells. (J) Summary of 5 experiments, which demonstrated that transfer was predominantly unidirectional from CD38⁺⁺ plasma cells to CD3⁺ cells (t = 3.5; P < .007). (K) Both autologous and allogeneic CD3⁺ cells acquired a similar level of biotinylated proteins from CD38⁺⁺ flow sorted primary plasma cells.