Slices of human LNs were exposed to DiO-, or DiD-labeled liposomes for 0.5, 2, or 24 hours. Cells were mechanically dissociated for flow cytometry, or the tissue was prepared for microscopy. (A) Representative flow cytometry plots showing DiO-liposome uptake after 2 hours of bathing, by resident myeloid cells and migratory skin-derived cells (n = 3–5). (B) Immunofluorescence image of LN slice showing DiD-labeled liposomes penetrated the tissue within 30 minutes of exposure. Arrow indicates exposed face. Scale bar: 500 μm. n = 3. (C) Uptake of DiD-labeled liposomes over 24 hours, measured by flow cytometry (n = 3). Colors in C, E, and F correspond to the cell subset legend. (D) Uptake at 2 hours was compared between LN cell subsets of the major groups: macrophages/monocytes, migratory DCs (Mig. DCs), resident DCs (Res. DCs), and lymphocytes. The median and IQR are plotted for each subset. Mixed-effects analysis with Tukey’s multiple-comparison test was performed. *P < 0.05 and **P < 0.01. For grouped statistical representation, the highest common P value is presented, but lower values were generated. (E) Immune cell subsets present in the LN (n = 50) and (F) making up the total liposome⁺ fraction after a 2-hour exposure (n = 5), showing cell subsets as a proportion of total live, CD45⁺ immune cells and myeloid cell subsets as a proportion of HLA-DR⁺ cells. (G) Mass cytometry image showing CD169 (red) and CD68 (green) staining in the LN. CD169⁺CD68⁺ SMs appear yellow; DAPI staining is shown (blue). Scale bar: 200 μm. (H) CD169⁺ (red) subcapsular SMs and CD11c⁺ (blue) DCs took up DiD-labeled liposomes (green) in situ in the LN after a 2-hour exposure (indicated by arrows). The capsule is visible at the top right. Scale bars: 25 μm. The median with the IQR for available donors is shown for each cell subset at each time point.