Multistep purification process provides highly purified memory B cells, CD4⁺ T cells, and CD11c⁺ DCs. (A) Highly purified memory B cells and CD4⁺ T cells were obtained from spleens of hemophilic mice treated with 4 weekly doses of 200 ng FVIII. After initial depletion of unwanted cells by magnetic cell separation, wanted cells were purified by flow cytometric single-cell sorting. Presented is the gating strategy (i-iv) for the purification of IgM⁻IgD⁻CD138⁻CD11c⁻CD19⁺ memory B cells and CD4⁺ T cells (dump: remaining unwanted FITC⁺ cells after initial magnetic cell separation). A representative result of the purity of sorted cell populations (gated on viable cells) is shown in panel vi. (B) Highly purified CD11c⁺ DCs were obtained from spleens of naive hemophilic mice. After initial depletion of total T cells and other unwanted cell populations using a 2-step magnetic cell separation, CD11c⁺ DCs were further purified by flow cytometric single-cell sorting. Shown is the gating strategy (i-v) for the purification of CD11c⁺ DCs (dump: remaining unwanted cells after initial magnetic depletion). A representative result of the purity of sorted CD11c⁺ DCs (gated on viable cells) is shown in panel vi.