Calcium flux assay
Purified B cells were incubated with 0.5 μM calcium-sensitive dye Fluo-4
AM (S1060, Beyotime) at 37 °C for 25 min and then stained with anti-B220
Ab for 30 min. Using a LSRII flow cytometer
(BD Biosciences, Franklin lakes, NJ,
USA), a baseline fluorescence was recorded for the first 30 s and then
cells were immediately stimulated with 10 μg/ml pre-warmed
biotin-conjugated F(ab’)2 anti-mouse Ig (M + G). The
fluorescence intensity was monitored for the next 300 s. Calcium flux
kinetics were analyzed using the FlowJo 7.6 software.