Differentiation and Stimulation of Murine Dendritic Cells and Macrophages

Bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs) were differentiated from bone marrow cells, obtained from the mice detailed above, as previously described [29 (link)]. In brief, cells were grown in complete RPMI 1640 with GlutaMAX (Gibco) with 10% FCS (Biowest), 1mM sodium pyruvate, HEPES 10mM, MEM non-essential amino acids, 2Mercaptoethanol 0,05mM and gentamicin 0,02mg/ml (all from Gibco). BMDCS and BMDMs were differentiated with GM-CSF from J558L cell line supernatants [30 (link)] or M-CSF from L929 fibroblast cell line supernatants, respectively. At day 8 and 7, respectively, BMDCs were 80% CD11c+CD11b+ and BMDMs were 95% CD11c-CD11b+. Total cells were harvested and cultured in 6-well plates (3x10⁶/well) and stimulated for 6 h with killed or live L. major promastigotes at a parasite/cell ratio of 5:1, or 250 ng/ml of CpG 1826 or 100ng/ml of conventional LPS (E. coli, O111:B4, Sigma). Cells were harvested at 6h after stimulation for RNA extraction. At 24h, cell-free supernatants were harvested for cytokine determination by specific ELISA.