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).