Figure 4.
Exosomes production by D1 cells upon maturation. (A) Exosomes were purified from a 24-h supernatant of D1 cells in control culture medium (control), or in the presence of 20 μg/ml LPS (LPS 24h), or in the presence of LPS and after a 16-h pretreatment with LPS (LPS 40h). The same number of D1 cells was used in each condition. Exosome production in the presence of LPS, as quantified by the Bradford assay, was rapported to the production by control cells. This graph represents the average of seven (LPS 24h) or two (LPS 40h) independent experiments. LPS treatment reduces the production of exosomes by D1 cells. (B) D1 cells in control conditions (Cont) or after 40 h in the presence of LPS (LPS 40h) were analyzed by FACS® after staining with antibodies specific for MHC class II molecules (MHC II) and CD40 (grey histograms; white histograms are negative controls obtained without primary antibodies), or after 15 min internalization at 37°C of FITC-coupled 40,000-Dalton dextran (DX-FITC) (grey histogram; white histogram is a negative control obtained after 15 min internalization at 4°C). As expected from mature D1 cells, both MHC class II and CD40 are upregulated at the surface of LPS-treated cells, whereas their endocytosis ability (as measured by the capacity to internalize dextran) is reduced compared with control cells.