Anti biotin magnetic microbeads

Enrichment of DC populations was performed by depletion of T and B cells from a cell suspension of dissociated spleen. Cells were incubated with 0.25 μg/ml biotinylated anti-CD19 (B cells: eBioscience; San Diego, CA, USA) and 0.2 μg/ml biotinylated anti-Thy1.2 (T cells: eBioscience) antibody per 10⁸ cells in 1 ml. Antibody was diluted in MACS labelling buffer (PBS/2 mM EDTA/5% BSA) and absorbed to cells for 10 min. on ice Cells were washed twice with labelling buffer before addition of anti-biotin magnetic microbeads (Miltenyi Biotec, Auburn, CA, USA). Cells were absorbed with 13 μl beads/10⁸ cells in 1 ml for 25 min. on ice. After washing with MACS labelling buffer, cells were resuspended in 500 μl of buffer. The cell suspension was then run through a pre-washed LS column (Miltenyi Biotec) positioned in the strong magnetic field of a SuperMACS II Separator (Miltenyi Biotec), which retains cells with bound magnetic microbeads. Splenocytes depleted of T and B cells were collected as flow-through cells, along with cells collected after three column washes with 3 ml labelling buffer. These cells were resuspended in sDMEM for staining.

Total spleen cells were obtained and depleted of B and T cells (in order to enrich for donor CFSE-labeled cells) by MACS using biotinylated anti-CD3 and anti-CD19 antibodies and anti-biotin magnetic microbeads (all from Miltenyi Biotec). The remaining cells were stained for CD11b and c-Kit, and analyzed by flow cytometry.