Flt3l

Bone marrow was RBC lysed and cells seeded at 1 × 10⁶ cells/ml in 6-well tissue culture plates in RPMI-1640 supplemented with glutamax, 10% heat-inactivated FBS, β-mercaptoethanol (5 0 μM), penicillin/streptomycin (100 μg/ml), and HEPES 1 mM (Sigma) and Flt3L (200 ng/ml; Biolegend). Flt3L-BMDCs were cultured for 8 days at 37°C and 5% CO_2. At day 8 non-adherent and adherent Flt3L-BMDC were harvested with trypsin-EDTA (Sigma) incubated for 2 min at room temperature. Cells were washed and live cells counted by trypan blue discrimination and seeded at 1 × 10⁶ cells/ml on 96-well flat bottom plates for 48 h in the presence or absence of anti-LTβR (2 μg/ml; 3C8; AdipoGen).

ES cell-derived HPCs were derived according to the protocol described previously by Chan et al. [3 ]. Briefly, HOXB4-transduced mouse ES cells (HM1 cell line) originally derived from the 129SvJ mouse strain were grown on feeder-seeded gelatinized flasks in ES cell culture medium with 15% fetal calf serum, 1% penicillin/streptomycin cocktail (GIBCO), 0.1 mM l-glutamine, and 1000 U/ml leukemia inhibitory factor for the maintenance of pluripotency. The ES cells were then subjected to embryoid body (EB) formation. The EBs were trypsinized and dissociated into a single cell dispersion before being replated onto ultralow-attachment Petri dishes in defined medium containing StemPro34 base media plus nutrient supplement (Life Technologies/BRL) and various hematopoietic cytokines: mIL-3 (2 ng/ml), mouse stem cell factor (100 ng/ml; R&D Systems), mIL-6 (5 ng/ml), IGF-1 (40 ng/ml; Promega), Flt3-L (10 ng/ml), and dexamethasone (1 μM; Sigma-Aldrich). The cell cultures were kept in a hypoxic incubator containing 9% CO₂.

For the in vitro experiment, bone marrow-derived DCs were generated by cultivation of bone marrow cells in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin solution (Nacalai Tesque) and 100 ng/ml of human fms-like tyrosine kinase 3 ligand (Flt3L) (PeproTech) for 7 days. Flt3L-induced DCs (FL-DCs), stimulated for 15 h with 1 mg/ml alum, 0.1% Endocine or 50 ng/ml LPS (Sigma), were evaluated for CD86 expression on plasmacytoid DCs (pDCs) by FACS. CD11c⁺/SiglecH⁺ cells defined as pDCs were used for analysis.
In vivo experiments were performed as described previously43 (link). Briefly, mice were injected with either 0.67 mg alum, 2% Endocine or 50 ng LPS to the base of the tail. Inguinal lymph nodes (iLNs) were collected from the mice 24 h after the administration of either adjuvant. To prepare single-cell suspensions, the iLNs were treated with DNase I (0.1 mg/ml) and collagenase D (1 mg/ml) for 30 min at 37 °C. Cells prepared by this way were stained with anti-mCD11c (HL3), -mPDCA-1 (JF05-1C2.4.1), -mCD86 (GL1) antibodies and 7-amino-actinomycin, and then analyzed by FACS. CD11c⁺/mPDCA-1⁺ cells defined as pDCs were used for analysis.