Validation of the flow cytometry method to quantify blood IPCs.

(A) Total viable PBMCs were gated based on their forward and side scatter (left panel). After a 2-color staining with anti–CD4-PE and (CD3, CD14, CD16, CD20, CD11c)-FITC, IPCs were identified as CD4⁺FITC⁻ cells (right panel). CD4⁺ T cells were identified as CD4^highFITC^high cells (right panel). For the IPC quantification, 10⁵ PBMCs were analyzed and the percentage of cells was determined by using the quadrant-stat function (CellQuest). For purification of IPCs, CD4⁺FITC⁻ cells were sorted. (B) Purified IPCs display a typical plasmacytoid morphology on cytospin preparation after Giemsa staining (×100). (C) Purified IPCs produce high amounts of type I IFN after 24 hours' stimulation with herpes simplex virus-1 as compared to cultures in medium alone.