Cd4 cd25 treg cell isolation kit

Spleens were homogenized and then passed through a 40 μm sterile filter (BD, UK). To isolate kidney resident lymphocytes, kidneys were flushed with PBS, homogenized, and then passed through a 40-μm sterile filter. Lymphocytes were isolated over a density gradient (Sigma), washed, and re-suspended in complete media. Cells were cultured at 1.25 × 10⁶ SMCs/mL in complete media consisting of MEM-α medium (Invitrogen) supplemented with 5% FCS, 4 mM l-glutamine, 100 μg/mL penicillin/streptomycin, 1 × nonessential amino acids, 1 mM sodium pyruvate, 50 mM 2-ME with appropriate stimulus for 5 days in a humidified atmosphere of 5% CO₂. Where required, Treg-phenotype cells and residual Teff cells were fractionated from SMCs with the CD4⁺CD25⁺ Treg-cell isolation kit (Miltenyi Biotech), and each fraction set at 0.5 × 10⁶ cells/mL, either alone or together, for co-culture with BM-derived dendritic cells as a source of APC at 1.25 × 10⁶ cells/mL. To generate BM-derived dendritic cells, the femurs and tibia from healthy mice were flushed, BM cells washed, and then incubated for 10 days in complete medium supplemented with 2.5% Ag8653 GM-CSF-containing supernatant (a gift from Brigitta Stockinger, NIMR, UK). Loosely adherent and nonadherent DC were recovered and enumerated for co-culture with T cells.

Heparinized venous blood was obtained from Control and RA patients. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation on Ficoll-Hypaque (EuroClone). PBMC were incubated with mAb-conjugated MicroBeads to human CD4 (Miltenyi Biotec) and CD4⁺ T lymphocytes were purified by magnetic separation. Purity of separated CD4⁺ T cells was >90% as detected by cytofluorimetric analysis. CD4⁺CD25⁺ and CD4⁺CD25⁻ cells were purified from CD4⁺ T lymphocytes using the CD4⁺CD25⁺ Treg cell isolation kit (Miltenyi Biotec) according to manufacturer's instructions. Unless otherwise specified, cells were seeded in 48 or 96-well culture plates pre-coated with mAbs to CD3 plus CD28 (both 2 µg/ml) and activated for 30 min with soluble mAbs to CD3 plus CD28 (both 3 µg/ml) (ImmunoTools) in RPMI-1640+10% FCS (complete medium) at 37°C plus 5% CO₂. After activation cells were washed and stimulated as described below.

Cells from spleen and LNs were purified using CD4⁺ MicroBeads (Miltenyi Biotech, catalogue number: 130-049-201), the CD4⁺CD62L⁺ T-cell isolation kit (Miltenyi Biotech, catalogue number: 130-093-227) or the CD4⁺CD25⁺ Treg cell isolation kit (Miltenyi Biotech, catalogue number: 130-091-041) according to the manufacturer's instruction. Purity as determined by flow cytometry was over 95%.