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.