Material internalization from live donor cells by DCs in vivo. C57Bl/6 CD45.1 mice were injected intravenously with live DiI-stained, CD45.2⁺ EL4 cells. At different times, spleens were collected, dissociated, and labeled. (A) Absolute numbers of EL4 cells, labeled by CD45.2 mAb, recovered from the spleen. Data are mean ± SEM (2 experiments, duplicates). (B) Viability of the EL4 cells was assessed by fluorescein intensity after esterase enzymatic activity on FDA. (C) DCs were gated on CD11c and CD8α expression to visualize CD11cCD8α⁺ and CD11cCD8α⁻ populations, and analyzed for DiI expression. Data are representative dot plots of cells analyzed 240 minutes after EL4 or PBS injection. (D) Association of DiI from the injected EL4 cells with CD8α⁺ or CD8α⁻ CD11c⁺ DCs. At 240 minutes later, spleen cells were labeled as described. Data are mean ± SEM (representative of 2 independent experiments performed in duplicates). (E) Spleen DCs were sorted 240 minutes after EL4 cell injection, labeled for CD11c, and observed by confocal microscopy. Six confocal planes were observed to ensure that live cell fragments were inside DCs. (F) Confocal microscopy imaging of the interaction between a spleen CD11c-EYFP^hi DCs and a DiD-stained EL4 cell. A total of 25 × 10⁶ EL4 cells stained with DiD (red, membrane staining) and with calcein red orange (green, cytoplasmic viability staining) were injected intravenously into CD11c-EYFP^hi (blue) transgenic mice. Thirty minutes later, the spleen was collected, cut into 2 slices, maintained in viable conditions, and observed by time-lapse confocal microscopy. Different focal planes were analyzed to ensure visualization of the whole volume of the DCs (original magnification ×20). Data are representative of 2 independent experiments (supplemental Figure 2).