Goat f ab 2 anti humanfc5μ

Anti-BCR antibodies used to stimulate B cells on PMSs and in solution were goat anti-mouse Igκ F(ab’)₂(Southern Biotech) for mouse splenic B cells and goat F(ab’)₂ anti-humanFc5μ (Jackson Immunoresearch) for Ramos cells. The antibodies were biotinylated with 20-fold molar excess of NHS-LC-LC-biotin (Pierce) and labeled with either Cy3 or Cy5 Monoreactive dyes (GE Healthcare), or with AlexaFluor 405 or AlexaFluor 647 NHS esters (ThermoFisher) in Sodium Carbonate buffer. Excess dye was removed using Zeba 7K MWCO desalting columns (Pierce, ThermoFisher).

Model antigens used in solution or on PMS were goat anti‐mouse Igκ F(ab')₂ (Southern Biotech) for mouse splenic B cells and goat F(ab')₂ anti‐human Fc5μ (Jackson Immunoresearch) for Ramos and DG75 cells. The antibodies were biotinylated using EZ‐Link NHS‐LC‐LC‐biotin (Pierce) and conjugated to one of several fluorophores ‐ Cy5 Monoreactive dye (GE Healthcare), AlexaFluor 405 or AlexaFluor 647 NHS esters (Thermo Fisher) in Sodium Carbonate buffer, according to the manufacturer's instructions. Excess dye was removed using Zeba 7K MWCO desalting columns (Pierce, Thermo Fisher). Mouse transferrin (Rockland) was also fluorescently labeled and biotinylated for TfR internalization assays.