B cell proliferation
CFSE-labeled B cells (1x106cells/ml) were cultured alone or cultured at various ratios with sorted CD8 Treg and activated with 2ug/ml of anti-human CD40/TNFRSF5 antibody and 2.5ug/ml of CpG in AIM-V medium and incubated at 37°C for 5 days. CFSE labeled B cells were stained with anti CD19 PerCP antibody and isotype control. 10,000 cells were acquired (FACSCalibur, Becton Dickenson, San Jose, CA), CD19+ B cells were gated and analyzed by Flow Jo software.
for 14 days (for immunoglobulin production) in 96-well round-bottom plates by ELISA assay.