Cd43 apc

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.

The floating hematopoietic cells were collected after 12–18 days of differentiation. Adherent cells were incubated with collagenase B (Roche) for 2 h, followed by treatment with cell dissociation buffer (Gibco). The cells were passed through a 70-μm cell strainer and incubated with the following fluorochrome-conjugated anti-human antibodies for 1 h at 4 °C: CD31-PE (#555446), CD34-FITC (#555821), CD45-APC (#555485), and CD43-APC (#560198) (all from BD Biosciences). Dead cells were excluded based on staining with 7-aminoactinomycin D (BD Pharmingen, #559925). The expression of HSPC markers was measured using a FACSCanto^TMII flow cytometer (BD Bioscience), and the acquired data were analyzed with FlowJo software (Tree Star).

For analysis of B cell differentiation, 5 x 10⁴ transduced HSCs were transferred to flasks of OP9 stromal cells and co-cultured in B lymphoid-promoting medium (Alpha MEM supplemented with 20% FBS, 1% penicillin-streptomycin, 50 μM 2-mercaptoethanol, 10 ng/mL SCF, 10 ng/mL Flt3L and 10 ng/mL mIL-7 (PeproTech, Rocky Hill, NJ), and 250 ng/mL amphotericin B). Cells were harvested by trypsinization and resuspended in sorting buffer after 7 days of co-culture. Hardy fraction analysis of B cell differentiation was performed as previously described [35 (link)]. Briefly, cells were stained with 7-AAD (eBioscience) and the following antibodies: B220-Pacific blue, CD43-APC, BP-1-BV605, CD24-PE-Cy7, IgD-APC-Cy7, and IgM-PE-CF594 (BD Biosciences). Single-color stained cells and UltraComp beads (eBioscience) were used for compensation and to establish Hardy fraction gating. The following controls were used to exclude OP9 cells and non-transduced cells from ZsGreen⁺/RFP⁺ analysis: OP9 cells alone, non-transduced HSPCs alone, and OP9 cells with non-transduced HSPCs. Samples were analyzed on an LSR II flow cytometer and FACS plots were generated using FlowJo software.