F ab 2 anti human igm igg iga

CD20⁺ B cells from patients and donors were purified from frozen PBMCs thawed in RPMI and 1% FBS plus 10 μL/mL DNAse (Calbiochem, San Diego, Calif). Purity was greater than 74%. CD20⁺ B cells were labeled with 0.5 μmol/L CFSE for 8 minutes at room temperature. CFSE-stained B cells (6 × 10⁴) from patients undergoing HSC-GT and donors were plated in 96-well flat-bottom culture plates and stimulated for 72 hours in RPMI 10% Hyclone (Sera; Thermo Scientific, Uppsala, Sweden) with 2.5 μg/mL CpG2006 (5′-TsCsg sTsGsg sTsTsT sTsgsT sCsgsT sTsTsT sgsTsC sgsTsT-3′; TIB BioMol, Genoa, Italy), 2.5 μg/mL F(ab′)₂ anti-human IgM/IgG/IgA (Jackson Immunoresearch), and 3 ng/mL CD40L (Alexis Biochemicals, San Diego, Calif). For patients undergoing ERT, 10 × 10⁴ CFSE-stained B cells were plated. Cell culture was performed in a final volume of 200 μL. The group of control subjects is composed of pediatric and adult donors who were verified to proliferate in the same way after stimulation. Proliferating B cells were stained with anti-CD19 PeCy7 and 7-amino-actinomycin D to exclude dead cells and IgG and IgA APC at the third day of stimulation. A BD FACSCanto II was used to acquire 10,000 events for each condition in the lymphocyte gate. The results were analyzed with FlowJo software 2.2.