Megakaryocytes produce APRIL and IL-6 in vivo. (A) Bone marrow megakaryocytes were identified by flow cytometry according to CD41 staining (left) and propidium iodide incorporation (middle, as a measure for polyploidy). Propidium iodide content of total bone marrow cells (dashed line, 2n-stadium) was used as a standard. Polyploid CD41⁺ megakaryocytes (n > 2) were stained for IL-6 (solid line) or isotype control (IL-4, dashed line). (B) Dot plot showing IL-6 or isotype control (IL-4) versus CD41. (C) Megakaryocytes, B cells, and macrophage cells were isolated by FACS, and IL-6 and APRIL expression was determined by quantitative PCR as indicated. MK indicates CD41⁺ megakaryocyte; BC, B220⁺ B cell; Mac, CD11b⁺/F4/80⁺/Gr-1⁻ macrophages; and Tot, total bone marrow cells. (D) Megakaryocytes were identified on bone marrow sections (femur) by CD41 staining (green) and counterstained for either APRIL or IL-6 (red) as indicated. Controls for IL-6 and APRIL stainings are shown in supplemental Figure 4.