Functional analysis of C1q produced by DCs and phagocytosis experiments. (A) The hemolytic activity of C1q present in DC culture media was investigated using SRBCs and supernatants derived from immDCs in culture (days 7-9). The amount of C1q quantified in ELISA was, respectively, 6.4 ng/mL (purified C1q), 339 ng/mL (DC I), 103.5 ng/mL (DC II), and 50 ng/mL (DC III). Results are shown as mean ± SD of triplicate analysis of DC supernatants from 3 different donors. *P < .001 compared with medium (ANOVA with Bonferroni correction). (B) 48-hour immDC supernatants were incubated for 2 hours with apoptotic Jurkat cells. Purified C1q (5 μg/mL) and fresh DC medium were used as controls. Binding of C1q was detected by flow cytometry using the mAb 2204. (C) CFSE-stained apoptotic cells were opsonized with purified C1q in serum-free medium overnight. After extensive washing, apoptotic cells were coincubated with immDCs for 2 hours each at 37° C and 4° C (DC/apoptotic cell, 1:1 ratio). As control, apoptotic cells cultured in medium alone were used. ImmDCs were stained with anti–HLA-DR. FACS analysis was performed considering the percentage of HLA-DR⁺/CFSE⁺ cells. Data are presented as mean ± SD of an experiment performed in triplicate. *P = .0072 compared with nonopsonized apoptotic cells (Student t test). Similar results were obtained in 4 experiments.