Fig. 2.

Phenotype of dendritic-like cells produced in co-cultures. Lin⁻ BM from C57BL/6 J mice was overlaid on 5G3 stroma to establish co-cultures and non-adherent cells were collected over time for staining with a range of antibodies for flow cytometric assessment of marker expression. (A) The CD11b⁺CD11c⁺ population of dendritic-like cells produced in 21-day co-cultures was gated for further analysis of marker expression. L-DC and cDC-like cells were distinguished through expression of MHC-II, whereby L-DC are CD11b⁺CD11c⁺MHC-II⁻ cells and cDC-like cells can be distinguished as CD11b⁺CD11c⁺MHC-II⁺ cells. Bivariate analysis of MHC-II expression with CD115, Sirp-α, F4/80, 4-1BBL, B220 and CD8α, served to identify the phenotype of the 2 subsets. (B) Non-adherent cells collected from 21 day co-cultures were incubated with FITC-ovalbumin (OVA) at 37 °C, or at 4 °C as control, to assess capacity of cells to endocytose antigen. At the end of incubation, cells were stained for markers, and L-DC and cDC-like subsets gated on the basis of phenotype as CD11b⁺CD11c⁺MHC-II⁻ and CD11b⁺CD11c⁺MHC-II⁺ cells, respectively. These were then assessed flow cytometrically for endocytosis in terms of % cells taking up FITC-ovalbumin (OVA) (red histogram) compared with controls (blue histogram)