Ficoll paque density gradient

PBMC were isolated by centrifugation on a Ficoll-paque density gradient (1.077 g/L, GE Healthcare. Chicago, IL, USA). Harvested LNs were washed with sterile PBS^+/+, cut in small pieces, and incubated in 15 ml of pre-warmed PBS^+/+ containing 0.36 mg/ml collagenase D (Sigma-Aldrich) and 100 µg/ml DNase I (Sigma-Aldrich) during 30 min under agitation at 37°C. The enzymatic reaction was stopped by adding 50 ml of PBS^-/- with 5 mM EDTA solution. The cells were then filtered through a 100 µm and 70 µm filter (BD Biosciences, Allschwil, Switzerland), washed three times with PBS^-/- (4°C). Cell count and viability was done with Türk’s solution under the microscope.
Isolated PBMCs or LN cells for T cell and B cell restimulation assays (1 x 10⁶ cells/ml) were cultured in DMEM/10%FBS with 1 µg/ml recombinant HA/NP (Immune Technology Corp., New York, U.S.A.), 1 µg/ml recombinant E2 [produced in cultures of insect cells infected with the baculovirus vector (42 (link))], or without (unstimulated). As a positive control, the cells from all samples were cultured with 10 µg/ml of Concanavalin A (Con A) (Sigma). Importantly, those experiments were performed in the absence of antibiotics. After 3 or 5 days at 39°C, cells were harvested and analyzed by flow cytometry (FCM).

Fresh PBMCs were isolated using Ficoll-Paque density gradient centrifugation (GE Healthcare, Uppsala, Sweden). PBMCs were cultured in RPMI media (Invitrogen, Carlsbad, CA, USA) supplemented with 4 mM L-glutamine and 10% FBS in the presence or absence of soluble anti-CD3 antibodies. A total of 250 ng of antibody was applied to 1 × 10 [6 (link)] PBMC/mL. T cells were isolated after 72 h of PBMC treatment, using magnetic beads by negative selection (according to the manufacturer’s instructions). Briefly, PBMCs were selectively depleted of CD16, CD19, CD20 cells and were discarded (Dynabeads® Untouched™ Human T Cells Kit, Invitrogen, Carlsbad, CA, USA). The purity of T cell enrichment was checked using flow cytometry (Additional file 1: Figure S1).

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood using Ficoll-Paque density gradient (GE Healthcare). PBMCs were washed with phosphate-buffered saline (PBS) and cultured in RPMI 1640 medium with 10 % FCS and 1 % glutamine/penicillin/streptomycin (Life Technologies). Recombinant human IL-33 protein was purchased from R&D Systems. Anti-human IgM antibody was purchased from eBioscience. Recombinant human CD40 Ligand (CD40L) was purchased from Life Technologies. For in vitro induction of anti-RBC IgG antibody, PBMCs (1 × 10⁶/ml) were cultured with anti-IgM (10 μg/ml) and CD40L (10 ng/ml) in the presence or absence of IL-33 (0–20 ng/ml). Six days later, the supernatants were collected and assayed for IgG antibodies with Human IgG total ELISA kit (eBioscience).