Rpmi 1640 complete medium

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy human donors by standard density-gradient centrifugation with Ficoll-Paque (Amersham Biosciences). CD14⁺ cells were purified from PBMCs by high-gradient magnetic sorting, using the VarioMACS technique with anti-CD14 microbeads (Miltenyi Biotec GmbH). Cells were then cultured in complete RPMI 1640 medium (Life Technologies) supplemented with 10 % (v/v) fetal calf serum (FCS), 20 ng/ml human GM-CSF (R&D Systems) and 10 ng/ml human IL-4 (R&D Systems) for 6 days (immature DC) (Chen et al., 2008 (link)). Human whole blood was obtained from healthy donors at the Taipei Blood Center of the Taiwan Blood Services Foundation, under a protocol (AS-IRB02–103202) approved by the IRB of the Clinical Center of the Department of Health, Taiwan. Written informed consent was obtained from all donors. For mouse bone marrow-derived DC (BMDC), bone marrow cells were isolated from femurs and tibias and cultured in RPMI 1640 complete medium supplemented with 10% (v/v) FCS, L-Glutamine, pen/strep and 40 ng/ml recombinant mouse GM-CSF (R&D Systems) for 9 days (Chen et al., 2008 (link)).

The isolated CXCR5⁺CD4⁺ or CXCR5⁺CD4⁻ T cells from five treatment-naïve PBC patients or four healthy controls were incubated with allogeneic CD19+ B cells from healthy controls in a 96-well plate, at a ratio of 1:1 in the presence of SEB (100ng/ml) in RPMI 1640 complete medium containing 10% FCS only or with rIL-21R-Fc (10 μg/ml; R&D Systems, Minneapolis, MN) or rIL-21 (20 ng/ml; Peprotech, Rocky Hill, NJ, USA) (30 (link), 31 (link)). After 7–8 days of culture, cells were harvested to evaluate surface phenotypes and supernatants were collected to detect IgM and IgG (32 (link)).