Isolation of CD8α⁺CD11c⁻Lin⁻cells from the spleen.

(A) The splenocytes enriched by CD8α-microbeads, as described in “Materials and methods,” were stained with biotin-conjugated anti-CD11c MoAb, followed by APC-conjugated streptavidin, as well as FITC-conjugated anti-CD8α, PE-conjugated anti-Lin markers (CD3ε, B220, Gr-1, CD11b, and NK1.1). The CD8α⁺Lin⁻cells for sorting were gated on CD11c⁻ cells. The purity of the CD8α⁺CD11c⁻Lin⁻ cells after sorting was presented (> 98%). The quads were set up on the isotype-matched control dot plot. The results are representative of more than 6 independent experiments. (B) CD8α⁺CD11c⁻Lin⁻ cells were first isolated by using a cell sorter and then further stained with PE-conjugated anti-Ia, anti-CD40, anti-CD86, anti-CD4, as well as biotin-conjugated anti-CD8β, revealed with APC-streptavidin. Solid and dotted lines indicated the immunofluorescence intensity of cells stained with a control and the test antibodies, respectively. The results are representative of 4 independent experiments. (C) CD8α expression of spleen-derived CD8α⁺CD11c⁻Lin⁻ cells. The CD8α⁺CD11c⁻Lin⁻ cells were first isolated by using a cell sorter as described in (A), and then reanalyzed by FACS (shown as solid line). For CD8αCD3ε T cells, the CD8α-microbead–enriched cells were stained with FITC-conjugated anti-CD8α and PE-conjugated anti-CD3ε. The CD8αCD3ε T cells were isolated by using a cell sorter and then reanalyzed by FACS (shown as dotted line). The results are representative of 3 independent experiments. (D) Giemsa staining was performed on CD8α⁺CD11c⁻Lin⁻ cells after sorting. The results are representative of 4 independent experiments. Original magnification × 400.