Different maturation stimuli inhibit the expression and production of C1q in mDCs. Day 7 immDCs were cultured for 48 hours in DC medium alone or with control L cells or in the presence of stimuli inducing maturation (LPS, TNF-α, or CD40L-transfected L cells). FACS was used to analyze cells for CD86 expression (A). Similar results were obtained for HLA-DR and CD83 expression (not shown). To detect mC1q, mAb 2204 was used (B). Supernatants were harvested (day 9) and tested for C1q using ELISA (C). All data shown are mean ± SD of triplicate analysis from a representative experiment with a single donor. LPS and TNF (*P < .0001) compared with immDCs; CD40L (*P < .0001) compared with L cell–immDCs (2-way ANOVA of 3 independent experiments). (D-E) At the end of 48-hour maturation experiments, mDCs and immDCs were collected, washed, and analyzed using flow cytometry. mDCs derived from culture with LPS, TNF-α (D), or CD40L-expressing cells (E) were then put in a secondary culture in medium only with GM-CSF as a survival factor without maturation stimuli. As a control, immDCs cultured in normal medium or derived from coculture with control L cells were used. Supernatants were harvested at indicated time points and tested for C1q. At the end of 24 hours (D) or 70 hours (E), cell viability was shown to be similar between immDCs and mDCs. Results are representative of at least 3 experiments, performed in duplicate or triplicate, with different donors.