Dexamethasone (dex)

Bone marrow was isolated from the femur and tibia from Balb/cAnNCrl (both male and female) 10–20 weeks old mice and seeded 450.000 fresh cells/ml in 6 wells plates (Corning costar). As culture medium IMDM (Gibco) supplemented with 10% FCS (Bodinco), 100 units/ml penicillin, 100 μg/ml streptomycin and 5 × 10⁻⁵ M β-mercaptoethanol in the presence of 20 ng/ml GM-CSF (in house produced) was used. On day 2 an equal volume of fresh culture medium containing 20 ng/mL GM-CSF was added, and on day 4/5 20 ng/mL fresh GM-CSF was supplemented to the culture. Tolerogenicity was induced by adding 10⁻⁶M dexamethasone (Invivogen) and 10⁻¹⁰M 1α,25-dihydroxyvitamin D3 (Enzo Life sciences) to the BMDC culture on day 7. Simultaneously with the tolerogenic agents, 10 μg/mL peptide (hPG: ATEGRVRVNSAYQDK) and maturation stimuli (lipopolysaccharide (LPS) 10 ng/mL; Sigma Aldrich or Monophosphoryl Lipid A (MPLA, 10 ng/mL) from Salmonella minnesota R595; Invivogen) were added. After 8 days of culture at 37°C, 5% CO₂, the BMDCs or tolDCs were harvested for further experimentation. Before co-culture, BMDCs or tolDCs were replated into 24 wells plates (Corning costar). Before injection in co-transfer experiments, BMDCs or tolDCs were thoroughly washed with medium (2x) and PBS (1x) and kept on ice.

Pharmacological agents were used in the following concentrations and time courses: cycloheximide (Calbiochem, 0.2 µg/µl, 2, 4, or 8 h), dexamethasone (Invivogen, 10 µM, 24 h).