Surface expression of mClec9A protein on DCs and other hemopoietic cells. (A) The DCs were purified and surface labeled by 4-color immunofluorescent staining. DCs were stained with mAb against CD11c (N418-PeCy7), CD45RA (14.8-APC), CD8 (53-6.7-APC-Cy7) and mClec9A (10B4-biotin). Splenic DCs were also stained with CD4 (GK1.5-FITC), thymic DCs with Sirpα (p84-FITC), and subcutaneous LN DCs with CD205 (NLDC-145-FITC). Splenic cDCs were divided into CD4⁺cDCs (CD11^hiCD45RA⁻CD4⁺CD8⁻), DN cDCs (CD11^hiCD45RA⁻CD4⁻CD8⁻) and CD8⁺cDCs (CD11^hiCD45RA⁻CD8⁺CD4⁻); thymic cDCs were divided into CD8⁻cDCs (Sirpα^hiCD8^lo) and CD8⁺cDCs (Sirpα^loCD8⁺); and LN cDCs into CD8⁻cDC (CD11c⁺CD205⁻CD8⁻), dermal cDCs (CD11c⁺CD205^intCD8⁻), Langerhans' cells (CD11c⁺CD205^hiCD8⁻) and CD8⁺cDCs (CD11c⁺CD205^hiCD8⁺), as described previously.³¹  pDCs were identified as CD11c^intCD45RA⁺. Splenocytes were stained with mAb against CD3 (KT3-1.1-FITC), CD19 (1D3-PeCy7), NK1.1 (PK136-PeCy7), CD49b (Hmα2-APC) and B cells (CD19⁺CD3⁻), T cells (CD19⁻CD3⁺) and NK cells (NK1.1⁺CD49b⁺CD3⁻) were identified. Splenic macrophages were enriched as indicated in “Isolation and analysis of Clec9A on mouse hemopoietic cells” and stained with CD11b (M1/70-Cy5) and F4/80-FITC and defined as CD11b^hiF4/80⁺. Bone marrow cells and splenocytes were stained with mAb against CD11b (M1/70-Cy5) and Ly6C (5075-3.6-FITC) and monocytes were defined as side-scatter^loLy6C^hiCD11b^hi. Bone marrow macrophages were Ly6C^intCD11b^hi. Cell populations were counterstained with SA-PE and analyzed for mClec9A expression. The solid line represents mClec9A staining on gated cells, the dotted line represents staining of the gated cells with an isotype-matched control. (B) Enriched preparations of splenic DCs were stained with mAb against mClec9A (10B4-biotin), CD11c (N418-Quantum dot 655), CD8α (YTS-169-PercpCy5.5) and CD24 (M1/69-Alexa 633) and 120G8-FITC, then counterstained with SA-PE. pDCs (CD11c^int120G8⁺) and cDCs (CD11c^hi120G8⁻) were analyzed for expression of mClec9A. mClec9A expression correlated with CD8α and CD24 expression on cDCs. Most splenic pDCs expressed mClec9A. (C) An enriched preparation of blood DCs was stained in parallel with the splenic DCs (B) using the same mAbs and analyzed using identical gating strategies. Blood DCs do not express CD8α, but do express CD24. Similar to splenic DCs, blood DCs expressing CD24 also coexpressed Clec9A. pDCs from the blood, like their splenic counterpart, expressed mClec9A.