Dual-color flow cytometry and cytospin preparations of sorted CD45+MHC IIhi cells from synovium-rich tissue (SRT) of hind paws. (a) Forward scatter (FS) and side scatter (SS) of light, showing a gate ('dendritic cell [DC] gate') based on the light scatter of DCs in rat pseudo-afferent lymph (a) and a gate containing the CaliBRITE beads used to calculate absolute numbers of cells (b). Cells expressing MHC class II molecules in SRT from a healthy rat (b-e) and from a rat 14 days after adoptive transfer of arthritis (f-i). All MHC II+ cells express CD45 (b, f). However, when CD45 events in (f) are plotted as a histogram (j), the MHC II- events (predominantly polymorphonuclear cells) express lower levels of CD45 than the MHC IIhi events (mononuclear cells). After adoptive transfer, more MHC IIhi cells express CD11c (c, g) and CD36 (e, i) but a minority express CD163 (d, h). Percentages indicate proportions of CD45+ cells that express indicated levels of MHC class II molecules (b, f) or MHC IIhi cells that express CD11c (c, g), CD163 (d, h), or CD36 (e, i). Morphology of CD45+MHC IIhi cells from SRT obtained 12 days after adoptive transfer of arthritis. The cells were sorted by fluorescence-activated cell sorting, and smears were prepared by cytospin (k-m). Giemsa stain shows that most cells have DC morphology (arrows), a few have veils (top inset), and a few (lower inset) have macrophage morphology (k). Indirect immunoperoxidase staining shows that cells with DC morphology (arrow and inset), but not macrophage morphology (arrowheads and inset), express CD11c (l). DCs did not express CD163 (arrows) but this antigen was expressed strongly by macrophages (arrowheads) (m). Objective, × 60. MHC, major histocompatibility complex.