Isolation of viable LCs by FACS. (A) Representative photomicrograph of the red pulp of a normal spleen stained with both anti-CD8α (brown) and anti-CD34 (magenta) antibodies (original magnification ×400; scale bar, 20 μm). (Right) Enlarged image of a part of the left panel. Arrows show LCs (brown) and SVECs (magenta). (B) The gating strategy for sorting SVECs and LCs. CD45⁺-cell depleted, DAPI⁻ viable single cells of collagenase B digested spleen specimens were first gated for CD45⁻CD3⁻ population (i). More than 99% of the CD45⁺ cell-depleted cells were CD45⁻CD3⁻. CD45⁻CD3⁻ cells were then gated for CD34⁺CD8α⁻ or CD34⁻CD8α⁺ populations (ii), which were then gated for CD45⁻CD3⁻CD34⁺CD8α⁻CD31⁺ SVECs (iii, light blue) and CD45⁻CD3⁻CD34⁻CD8α⁺CD31⁺ LCs (iv, magenta). Negative gates were determined according to corresponding isotype control staining. (C) CD31/CD8α FACS profiles of DAPI⁻CD45⁻CD3⁻ cells. (D) SSC/FSC FACS profiles of back gated LCs (magenta) and SVECs (light blue) from panel B. (E) CD34/CD206 FACS profiles of DAPI⁻CD45⁻CD3⁻ cells.