Generating Plasmacytoid Dendritic Cells

Bone marrow (BM) pDCs were generated from C5aR1^+/+ and C5aR1^−/− mice using a modified protocol previously described (26 (link), 27 (link)). Briefly, BM cells were harvested from mouse femurs and tibias. BM cells (2 × 10⁶) from C5aR1^+/+ and C5aR1^−/− mice were seeded in 6-well plates and cultured with RPMI 1640 medium containing 10% fetal calf serum, streptomycin (100 μg/mL), penicillin (100 U/mL), 2 μM glutamine, 50 μM β-mercaptoethanol, and recombinant mouse FLT3L (200 ng/mL; eBioscience, San Diego, CA, USA). Half culture medium was replaced every 5 days. In the another experiment, BM cells from C5aR1^+/+ mice were simulated with FLT3L alone or the combination of FLT3L and recombinant mouse C5a (42 nM; Peprotech, Rocky Hill, NJ, USA), and medium group was used as the negative control. At day 10, detached cells were collected and analyzed the percentage of pDCs by FCM.