B220 percp

Spleens were removed from mice and single-cell suspensions were prepared by mashing the spleen using a 3-ml syringe plunger on a strainer (70 μM) and washing cells with PBS. Single cell suspensions were stained for flow cytometric analysis with anti-CD3-APC, anti-CD4-Pac Blue, anti-CD8-Pac Orange, Anti- B220-Per CP, anti-annexin-FITC, and anti PI-PE (BD Pharmingen, San Diego, CA). Blood and peritoneal fluid were also harvested and single-cell suspensions were prepared. Cells were stained with Gr-1-FITC, B220-Per CP, CD11b-APC, CD3-Pac Blue, CD11c-PE-Cy7; or CD4-Pac Blue, CD8-Pac Orange, Ly49D-FITC, NK1.1-PE, CD127-APC.
To measure production of cytokines on a per cell basis, splenocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 30 ng/mL) and ionomycin (400 ng/mL) in the presence of 10 μg/mL of Brefeldin A. After 18 hours, cells were surface stained with anti-CD4 and anti-CD8 and processed with an intracellular staining kit (BD Biosciences) according to manufacturer’s instructions. Intracellular antibodies included anti-interferon (IFN)-γ (eBioscience, San Diego, CA), anti-TNF, and anti-IL-2 (both BD Biosciences). Data were acquired on a LSR II multicolor flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).

Ten-week-old WT and KO mice were weighed and anesthetized by intraperitoneal injection of pentobarbital sodium. Blood was collected from the internal canthus vein, EDTA anticoagulation. The blood count was used to determine the peripheral blood count and perform classification. Immune cells from peripheral blood were incubated with labeled antibodies. The NK1.1-FITC fluorescent-labeled antibody was purchased from Biolegend; CD11b-APC was purchased from Invitrogen; B220–PerCP, F4/80–PE, CD4-PE-Cy, and CD8a-APC-Cy7 were purchased from BD; B220–PerCP-Cy5.5 and CD3-APC were purchased from Biolegend Division; EDTA was purchased from Sigma; and Erythrocyte lysate was purchased from Invitrogen. We then added the cells to TruCOUNT tubes (BD Biosciences) and counted them on an LSR II flow cytometer (Becton Dickinson), before analyzing with FlowJo software (version 10).

Cells were incubated with Fc block (anti-CD16/32, BD) and stained with different panels of fluorochrome-conjugated monoclonal antibodies in PBS/2% FBS using the following antibodies: CD5 BV421 (S3-7.3), CD19 PE (1D3), CD19 FITC (1D3), CD19 BUV395 (1D3), CD43 APC (S7), B220 AF-700 (RA3-6B2), B220 PerCP, CD11b BV605 (M1/70) (all BD), CD93 APC (AA4.1), CD93 PE (AA4.1), B220 APC-EF780 (RA3-6B2), IgM eFlour450 (II/41) (all eBioscience), CD5 biotin (Biolegend), and IgM FITC (Fab₂, polyclonal) (Southern Biotech). In some antibody panels, primary staining was followed by addition of streptavidin-AF488 (Life technologies). Samples were run using a BD LSRFortessa or LSR II and data were analyzed in Flowjo v9.6.4 (Treestar).

Single-cell suspensions of spleen and LN cells were prepared as previously described [35 (link)]. Joint tissues from hind paws of KSTA mice were dissected free of soft tissue and bones, digested with 100 μg/ml Liberase (Roche) for 45 min at 37^oC, and filtered through a 70-μm-pore cell strainer (SPL Life Sciences) to prepare single cell suspensions of synovial infiltrates. The single cell suspensions were stained with an appropriate combination of mAbs and analyzed by FACS. The mAbs used were: CD138-PE, B220-PerCP or -APC-cy7, FAS-PE, CD19-PerCP or -APC, CD21-FITC or -APC, CD43-APC, CD23-PE-cy7, CD8a-PE, CD25-APC-cy7, CXCR5-biotin, GL7-FITC, CD45.2-FITC, CD27-FITC, Ki-67-FITC, Gr-1-FITC, CD4-FITC or -APC-cy7, phospho-Syk-PE, CD11b-PE or -PerCP, PD-1-APC, CD44-APC-cy7, and F4/80-PerCP mAbs (all from BD Biosciences, eBioscience or Biolegend). Streptavidin-PerCP was purchased from BD Biosciences.