Macs negative selection system

CD4⁺ T cells were isolated from spleens using the MACS negative selection system (Miltenyi Biotec), by staining with biotinylated antibodies (Abs) against CD19 (6D5), CD8a (53–6.7), CD11b (M1/70), CD49b (DX5), I-A^b/d (25-9-17), and Ly-6G/Ly-6C (Gr-1, RB6-8C5) (BD Biosciences) followed by streptavidin-microbeads (Miltenyi Biotec). CD4⁺CD25⁻ cells were isolated by adding biotinylated anti-CD25 Ab (7D4) (BioLegend) to the first Abs. CD4⁺CD25⁺ cells were isolated using the MACS positive selection system by staining with PE anti-CD25 Ab followed by anti-PE microbeads (Miltenyi Biotec). Foxp3 was expressed in 96% of the CD4⁺CD25⁺ population. Purified CD4⁺ T cells, CD4⁺CD25⁻ Tc cells, or CD4⁺CD25⁺ Treg cells from WT mice were adoptively transferred into syngeneic nu/nu or TCRα^−/− mice by intraperitoneal injection (3.0 × 10⁶ cells/mice). In some experiments, donor T cells were labeled with CFSE, as previously described7 (link). Briefly, cells were incubated with 5 μM CFSE for 10 min at 37 °C. B cells were positively isolated using the MACS system by staining with biotinylated anti-CD19 Ab (1D3) (BD Biosciences) followed by streptavidin-microbeads.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood using Lymphoprep™ (Axis-Shield PoC AS, Oslo, Norway) density gradient centrifugation. Subsequently, CD14⁺ monocytes were separated from PBMCs using a MACS® negative selection system (Miltenyi Biotech, Bergisch Gladbach, Germany) after the cells were pretreated with an Fc Receptor Blocking Reagent (Miltenyi Biotech) according to the manufacturer’s instructions. To further remove residual T cells, the cells were subsequently subjected to MACS® column separation using anti-CD3 monoclonal antibody (mAb)-coupled magnetic beads (Miltenyi Biotech). The proportion of CD3⁻CD14⁺ cells in the purified monocyte fraction was confirmed by flow cytometry and was consistently > 97%.