Identification of donor-derived CD45⁺ leukocytes by flow cytometry. (a) Representative sequential gating strategy for the identification of CD45⁺ leukocytes in human peripheral blood, liver tissue, and hepatic hilar lymph nodes (live, singlet cells) using 16-color flow cytometry. FSC-A, forward scatter; SSC-A, side scatter; Live/dead, fixable dead cell stain. (b) Two representative flow-cytometric plots showing identification of donor- and recipient-derived CD45⁺ leukocytes using HLA-class I monoclonal antibodies, gated using a fluorescence minus one (FMO) control. (c) Frequency of donor-derived CD45⁺ leukocytes (gated on live, singlet cells; n = 6; six independent experiments) identified in the allograft for each individual in relation to time to explant, based on monoclonal antibody staining for donor-derived HLA-class I haplotype exemplified in panel b. (d) Representative flow-cytometric plots showing donor and recipient HLA-class I haplotype staining exemplified in panel b on “classical” Lin⁻HLA-DR⁺CD14⁺CD16⁻ MNPs and DC subsets (Lin⁻HLA-DR⁺CD14⁻CD16⁻CD123⁺CD11c⁻ plasmacytoid DCs [pDCs], Lin⁻HLA-DR⁺CD14⁻CD16⁻CD123⁻CD11c⁺ conventional DCs [cDCs]) within an allograft.