Ramos B cell calcium flux assay

Ramos B cell calcium flux was measured as described (75 (link)). Protein tetramers were formed at a 4:1 molar ratio of protein to streptavidin (Invitrogen). Ramos cell lines stably expressing DH270 UCA, DH270.1, or CH65 IgM (76 (link)) were passaged (1:10) 4 days before calcium flux experiments. On the day of the experiment, cells with >95% viability were resuspended at 10⁶ cells ml⁻¹ in 2:1 ratio of RPMI media (GIBCO) + FLIPR Calcium 6 dye (Molecular Devices). Cells were plated in a U-bottom 96-well tissue culture plate (Costar) and incubated at 37°C 5% CO₂ for 2 hours. In a black clear-bottom 96-well plate (Costar) containing 50 μl of RPMI media (GIBCO) + FLIPR Calcium 6 dye (Molecular Devices) (2:1 ratio) either 0.1 nmol of proteins or 50 μg ml⁻¹ of anti-human IgM F(ab’)2 (Jackson Immuno) were added (based on a 100-μl volume). Using a FlexStation 3 multimode microplate reader (Molecular Devices), 50 μl of supernatant containing cells were transferred into the 50 μl of media containing protein or anti-human IgM F(ab’)2 (Jackson Immuno) and continuously read for 5 min. Relative fluorescent value units were background-subtracted and the data expressed as percentage of the IgM maximum signal (% IgM max).