Imag cell separation system

pDCs were isolated from PBMCs with BD IMAG™ Cell Separation System (BD Biosciences). PBMCs were first separated from human peripheral blood by using density gradient centrifugation and pDCs were obtained following a manufacturer’s protocol. pDCs were then seeded at 8 × 10⁵ per well in 200 μl of complete medium (RPMI 1640 medium supplemented with 10% FBS and 50 μg/ml Gentamicin). The cells were stimulated with either CpG alone or CpG together with differently citrullinated forms of LL-37. Both factors were mixed in PBS 10 min prior to cell stimulation at the indicated concentration: CpG (final concentration 3 μM) and LL-37 (final concentration 28.8 μg/ml) (molar ratio 1:1). Subsequently, cells were incubated with peptide-CpG mixtures for 15 min, washed with PBS 3 times and incubated in fresh complete medium overnight at 37 °C in a humidified 5% CO₂ atmosphere. Cell morphology was visualized by bright-field microscopy and images were taken, while culture supernatants were collected for cytokine measurement with ELISA.
RAW264.7 (0.2 × 10⁶ cell/ml) were seeded in 200 μl of DMEM supplemented with 10% FBS and 50 μg/ml PEST overnight at 37 °C in a humidified 5% CO₂ atmosphere and adherent cells were washed with PBS once on the next day before fresh medium was added. Thereafter, macrophages were stimulated with CpG mixed with peptides as described above for pDCs.