PMN-MDSCs and B cells isolation and culture assay
Splenocytes from CIA and DBA/1J mice were stained with anti-CD11b,
anti-Ly6G, anti-Ly6C, and anti-CD19 antibodies.
CD11b+Ly6G+Ly6ClowPMN-MDSCs and
CD19+B cells were sorted using FACS Aria II I sorter (BD Biosciences). The
purity of the sorted PMN-MDSCs and B cells was >95%.
1×10^5 CD19+ B cells per well were plated in
96-well U-bottom plates, stimulated in RPMI 1640 medium containing 10%
FBS,
50𝜇g/ml
anti-CD40 (FGK4.5, Bio X Cell) and 10ng/ml IL-4 (GenScript) at 37℃ and
5% CO2 for 48h (for apoptosis test) or 72h (for TNF-𝛂
and Ki67 test), and cocultured with PMN-MDSCs in different ratios or
alone. To block function of BAFF,
5𝜇g/ml
anti-mouse BAFF (R&D) were added. To test function of BAFF, 100ng/ml
mBAFF (R&D) were added in the B cell culture assay.