Biotinylated goat f ab 2 anti human λ κ

PLB was prepared in 8-well Lab-Tek chamber (#1.0 Borosilicate coverglass system, Nunc) as described before (29 (link)). Briefly, PLB was prepared using 110 μM small unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (DOPE-cap biotin) (Avanti Polar Lipids) in ratio 100:1. To bind Ag to the PLB, the wells containing PLB were incubated at RT with 2.5 μg/ml streptavidin for 10 min, followed by 1 μg/ml biotinylated goat F(ab′)₂ anti-human λ + κ (Southern biotech) for 20 min.
PMS was prepared in 8-well Lab-Tek chamber (#1.5 Borosilicate coverglass system, Nunc) as previously described (30 (link)). Briefly, 293A cells (1 × 10⁵) were seeded in poly-l-lysine-coated wells and cultured overnight in complete RPMI at 37°C, 80–90% confluency being achieved. Cells were washed with HBSS and sonicated with a probe sonicator (5 s, 22% power) in HBSS containing 2% BSA to obtain PMS. To bind Ag to the PMS, the wells containing PMS were first blocked with HBSS containing 2% BSA for 30 min at RT and incubated sequentially for 30 min with 1 μg/ml biotinylated annexin V (Biolegend), 1 μg/ml streptavidin and 0.5 μg/ml biotinylated goat F(ab′)₂ anti-human λ + κ (Southern biotech).
Ag concentrations for PLB and PMS were selected by titration measurements to contain the same amounts.

Tonsil B cells (1 × 10⁶) were stimulated with various combinations of BCR and TLR9 ligand, in presence of cytokines, IFN-γ and IL-12+IL-18. For BCR stimulation, 10 nM biotinylated goat F(ab′)₂ anti-human λ + κ (Southern biotech) was used for either soluble Ag or membrane-bound Ag on PLB or PMS. B cells were cultured in complete RPMI at 37°C for 40 h in 8-well chambers containing either PLB-Ag or PMS-Ag for membrane Ag engagement and B cells were cultured in round-bottomed 96-well plates for soluble antigen engagement. When needed, cells were supplemented with CpG (ODN2006) (1 μM, Invivogen) in culture media for TLR9 stimulation. For cytokines, rhIFN-γ (50 ng/ml, Biolegend), rhIL-12 (50 ng/ml, Biolegend), and rhIL-18 (50 ng/ml, MBL) were used.