Sorting and proportion of FMDV serotype A-specific B cells via different bait antigens by flow cytometry.
Bovine PBMCs (A) were analyzed by 3-color flow cytometry and the gate P1 (B) was selected to exclude cells debris with lower values of SSC-A and FSC-A, and further analyzed singlets in gate P2 (C) based on diagonal streak of the FSC-A and FSC-H plot. The class-switched (IgM-) B cells in gate P3 were used to check the distribution of CD21 and FMDV-specific cells, in the presence of FMDV bait antigen A/GDMM/2013 (D) and A/AF72 (E), respectively. The demarcation line between FMDV-specific binding and nonbinding B cells was set according to the FMO control (F) in absence of bait antigen. The CD21+/- Antigen+ B cells in gate P4 were separately sorted for preparation of bovine mAbs. Appropriately one million bovine PBMCs were collected to analyze the distribution proportion of A/GDMM/2013-binding (G) and A/AF72-binding (H) B cells in circulating peripheral blood.