Flow cytometric analysis
Single-cell suspensions were prepared from the spleens and joints of mice. After incubation with anti-CD16/CD32 (93, Biolegend) for 20 min at 4℃, dead cells were excluded with eBioscienceTMFixable Viability Dye eFluorTM 506, and then anti-mouse mAbs against Gr-1(RB6-8C5), Ly6G(1A8), CD11b(M1/70), Ly6C(HK1.4), CD19(6D5) were used to stain specific surface antigens for 20 min at 4℃. For intracellular cytokine and nuclear factor staining, single cell suspensions were cultured at 37℃ for 4h in RPMI 1640 media containing 100 ng/ml phorbol-12-myristate-13-acetate (Enzo), 1 𝛍g/ml ionomycin (Enzo) and 5 𝛍g/ml brefeldin A (Enzo). Cells were fixed, permeabilized, and stained with TNF-𝛂 (MP6-XT22) and Ki67 (16A8) after staining with CD19. Abs were conjugated with APC, FITC, PE, PE-cy7, or PE/DazzleTM 594 from Biolegend. Data were acquired using BD LSRFortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star).