Pdc isolation kit

Peripheral blood mononuclear cells (PBMCs) were purified from healthy blood donor buffy coats or EDTA-blood from SLE patients and healthy donors using Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation. PDCs and B cells were isolated from PBMCs by magnetic bead separation using negative selection (pDC Isolation kit and B cell Isolation kit II; Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of the isolated cell populations was determined by flow cytometry after staining with anti-BDCA2 (Miltenyi Biotec) or anti-CD19 (BD Biosciences) monoclonal antibodies (mAbs) and was found to be at least 95%. The pDCs and B cells were cultured as previously described with 0.25x10⁵ pDCs and 1x10⁵ B cells in 0.1 ml volumes per well in 96-well plates for 20 hours or 4 to 6 days as indicated, at 37°C with 5% CO₂ [17 (link)]. When indicated, affinity purified polyclonal goat anti-BAFF (#AF124) or normal goat IgG antibodies (R&D Systems) were added to the co-cultures of pDCs and B cells at a final concentration of 20 μg/ml at the beginning of the cell culturing.

The pDCs were isolated from splenocytes using a pDC isolation kit (Miltenyi Biotec, Auburn, CA, USA). The pDC isolation purity was determined via flow cytometry, and the purity of CD317⁺B220⁺ pDCs was higher than 90%.