PRJNA486523
Mus musculus Genome sequencing
The cells extracted from the mice bone marrow were cultured in 6-well plates (2.5 × 105 cells/well) in 2 ml of complete DMEM high glicose (Gibco) supplemented with 10% FBS , glutamine, 2-mercaptoethanol, penicillin/streptomycin and 20 ng/ml GM-CSF. The plates were kept at 37oC at 5% CO2 and, after four incubation days, 1 ml of media was replaced by 1 ml of fresh DMEM supplemented with 40 ng/ml GM-CSF. By the seventh day, 500 µl of media was replaced by the same volume of fresh media without GM-CSF. On day 9, non-adherent cells and loosely adherent cells were harvested, stained in ice-cold PBS containing 1% BSA using anti-CD11c Monoclonal Antibody-PerCP-Cyanine5.5 (eBioscience) and sorted by FACS.Indirect co-culture of MoDC with Pb18 was performed using a transwell system (0.4-µm membrane porosity) in 1 ml DMEM supplemented with 10% FBS in each chamber. Pb18 yeast cells (1 x 106) were seeded in the upper chamber, and 1 x 106 CD11c+ cells (dendritic cells, MoDC) were placed in the lower chamber of a six-well plate. The plates were incubated for 48 h at 37oC at 5% CO2. The MoDCs were harvested, washed and used for RNA extraction.