Stemxvivo serum free dc base medium

Leukocyte apheresis cones were collected from healthy donors at the National Blood Service (Manchester, UK). PBMCs were separated by centrifugation using Ficoll-Paque (GE Healthcare, Amersham, UK). Monocytes were separated from PBMCs using anti-human-CD14 magnetic beads (Miltenyi Biotec, Cologne, Germany) according to the manufacturer’s instructions, using an LS MACS separation column. Monocyte purity was consistently over 95%.
Monocytes were cultured in StemXvivo serum-free DC base medium (Bio-techne, Minneapolis, MN, USA) containing 25 ng ml⁻¹ GM-CSF and 25 ng ml⁻¹ interleukin-4 (Biolegend, San Diego, CA) for 6 days at a concentration of 0.5 × 10⁶ cells per ml in 24-well tissue culture-treated plates. Half of the medium was removed on day 3 and replaced with fresh medium and cytokines. After 6 days of differentiation, cells were treated for 48 h with different combinations of compounds: 5 ng ml⁻¹ active TGFβ (Peprotech, Rocky Hill, NJ), 100 μg ml⁻¹ anti-TGFβ antibody (clone 1D11, West Lebanon, BioXcell, NH), 1 µM pan-RA receptor antagonist (LE540, a kind gift from Hiroyuki Kagechika, Tokyo Medical and Dental University, Tokyo, Japan), 100 nM RA (Sigma Aldrich, St Louis, MO, USA), 1 µg ml⁻¹ Pam3CSK4, 10 μg ml⁻¹ Poly(I:C), 1 µg ml⁻¹ flagellin, 5 μg ml⁻¹ Imiquimod, 5 μg ml⁻¹ ssRNA40, or 100 ng ml⁻¹ LPS (all from Invivogen, San Diego, CA).