Figure 3

Activation status of dendritic cells and platelets during co-culture. Dendritic cells (DCs) were cultured for 48 h alone (untreated), with sCD154, or with platelets (PLTs) either in two 0.4-μm pore-sized filter-separated compartments or in the same compartment. To evaluate the contribution of soluble factors released by PLTs on DC activation, PLTs were used either untreated or following fixation with paraformaldehyde. DCs used in co-cultures were untreated, preincubated with blocking anti-CD162 mAb to prevent PLT binding when placed in the same well, or preincubated with suramin in order to block ATP receptors. DCs were then stained for CD1a and CD80, CD86, CD83, or CD40 and analyzed by flow cytometry. The flow cytometry plots are gated on CD1a⁺ cells only. PLTs cultured without DCs (untreated), after thrombin activation, and after co-culture with DCs in separate or the same compartments were stained for CD41 and CD62P. (A) Representative results of five independent experiments and (B-D) mean values ± SD of three independent experiments for suramin, blocking anti-CD162 mAb, and fixed PLTs conditions, and of five independent experiments for the other conditions. Mean values for activation marker expression by DCs co-cultured with PLTs are summarized (B) and developed for co-culture in the same well (C) and for filter-separated co-culture (D). Percentages of marker expression in each experiment were assessed in triplicate. Asterisks indicates statistical significance between assay and untreated DCs a p < 0.05 (*) or < 0.005 (**) by Mann-Whitney U test.