F ab 2 fragments

For mouse in vitro activation studies, FO B cells and MZ B cells were sorted using the FACSAria or FACSAria SORP (BD). For dead cell exclusion, 20 ng/mL DAPI (Thermo Fisher Scientific) was added to the cells just prior to sorting. FO B cells and MZ B cells were cultured in RPMI medium plus penicillin-streptomycin, l-glutamine, and 10% FBS and stimulated with polyclonal goat anti–mouse IgM (Southern Biotech; 0.6–150 μg/mL), or with equimolar concentrations of the Fab′2 fragments (Southern Biotech; 0.4–100 μg/mL) for 20 hours, after which activation was measured using flow cytometry as described above.
For human in vitro activation studies, naive and MZ-like B cells were sorted using the BD FACSAria system. Cells were cultured in RPMI medium plus penicillin-streptomycin, l-glutamine, and 10% FBS and stimulated with polyclonal goat anti–human IgM (Jackson ImmunoResearch, AffiniPure; 0.6–75 μg/mL), or with equimolar concentrations of the Fab′2 fragments (Jackson Immunoresearch Affinipure; 0.4–50 μg/mL) for 20 hours, after which activation was measured using flow cytometry as described above.

HumAb agglutination of ST3 was determined by flow cytometry as described (23 (link), 55 (link)). ST3 strains A66 or B2 (1 × 10⁵ CFU) were incubated with humAbs, F(ab′)₂ fragments, or human IgG1 (control) (Southern Biotech) for 1 h at 37°C in a 96-well plate. Cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry. Bacteria were gated on forward scatter (FSC) and sideward scatter (SSC) (referring to cell size and granularity) to determine percent agglutination. Agglutination was also assessed by light microscopy. Aliquots from each sample were spotted onto 1% agarose pads and visualized with an AxioImager Z1 microscope (Zeiss).

HumAb agglutination of ST3 was determined by flow cytometry as described (23 (link), 55 (link)). ST3 strains A66 or B2 (1 × 10⁵ CFU) were incubated with humAbs, F(ab′)₂ fragments, or human IgG1 (control) (Southern Biotech) for 1 h at 37°C in a 96-well plate. Cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry. Bacteria were gated on forward scatter (FSC) and sideward scatter (SSC) (referring to cell size and granularity) to determine percent agglutination. Agglutination was also assessed by light microscopy. Aliquots from each sample were spotted onto 1% agarose pads and visualized with an AxioImager Z1 microscope (Zeiss).