CD4⁺ and CD8α⁺ DC present in fresh spleen suspensions.

Low-density spleen DCs were gradient-enriched by Nycodenz gradient centrifugation from spleen. At this stage DCs were not further purified by immunodepletion, but immediately labeled in 3 colors for CD11c (plus FITC-antihamster Ig; FL-1; not shown), the indicated monoclonal antibodies (mAb; plus PE-antirat Ig; FL-2) followed by blocking in 10% rat serum and addition of Cy5-PE–conjugated CD4 (FL-3). Plots show the expression of antigens on fresh DCs (A) or the effect of culture on the indicated antigens (B). Dot plots show DCs gated for CD11c positivity and by size and forward scatter to exclude dead cells and debris (see McLellan and Kampgen19).