Macs magnetic microbead kit

Pooled single cell suspensions of SPCs and lymph node cells (LNCs), herein referred to as peripheral blood mononuclear cells (PBMCs) were obtained from EAE mice 2 weeks after immunosuppressive therapy with MIC_Cop as described above. Regulatory CD4⁺CD25⁺ T lymphocytes (Tregs) were purified by consecutive negative isolation of CD4⁺ cells and positive selection of CD25⁺ lymphocytes using a MACS^® magnetic microbead kit as specified by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany), usually resulting in a purity above 90 % of the CD4⁺CD25⁺ cell population. Importantly, initial flow cytometric examination revealed that nearly all (>95 %) CD4⁺CD25⁺ lymphocytes also expressed the transcription factor FoxP3.

PB and BM were isolated and analyzed as previously described (61 (link), 71 (link), 72 (link)). Briefly, PB was collected in fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS), 2% fetal bovine serum, and 2 mM EDTA] supplemented with 0.0004% heparin solution (STEMCELL Technologies Inc.). RBCs were removed by incubation in 1% dextran solution in PBS at 37°C for 25 min. The cells in the supernatant were isolated and subjected to room-temperature RBC lysis solution (STEMCELL Technologies Inc.). PB cells were stained with conjugated antibodies targeting CD4, CD8, NK1.1, CD19, CD11b, Gr-1, CD45.1, and CD45.2. BM cells were isolated from tibias, femurs and hip bones that were crushed with a pestle and mortar. Lineage depletion (B220, CD4, CD8, CD11b, Gr-1, and Ter119) or c-kit enrichment was performed using MACS magnetic microbead kits (Miltenyi Biotec) according to the manufacturer’s instructions. BM cells were stained with conjugated antibodies against B220, CD4, CD8, CD11b, Gr-1, Ter119, cKit, Sca1, CD48, CD150, CD45.1, CD45.2, CD105, CD41, CD16/32, and MHC class II. Streptavidin-BV510 was used for biotin identification. Propidium Iodide (1 μg/ml; Molecular Probes) was used to distinguish viability. Flow cytometry analysis and assisted cell sorting were performed using Beckman Coulter CyAn ADP, Becton Dickinson (BD) LSRFortessa X-20, and BD Aria III.