Differentiation of Myeloid Dendritic Cells

Mice were euthanized by overdose of the association of dissociative anesthetic (ketamine, 300 mg/Kg) and alpha 2 adrenoreceptor agonist (xylazine, 30 mg/Kg) administered intraperitoneally. Bone marrow precursors were collected from the tibia and femur, and the cell suspension was adjusted to a concentration of 2 × 10⁵ cells/mL and seeded in a 96-well plate. Cells were maintained for seven days (culture medium was changed every other day) at 37 °C and 5% CO₂ in Iscove’s Modified Dulbecco’s Medium (IMDM), containing 10% fetal bovine serum (FBS), 50 μg/mL gentamicin (Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA) and supplemented with 10 ng/mL Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF; Biolegend, San Diego, CA, USA; #505406) [17 (link)]. This protocol differentiates precursor cells in conventional/myeloid DCs. Supplementary Figure S1 shows the gate strategy and percentages of DCs obtained from precursor cells.