Immunization-induced plasma cells colocalize and interact with megakaryocytes. (A) Distribution of Ova-specific plasma cells and megakaryoctes in spleen and bone marrow. Numbers of total CD138⁺ plasma cells (PC), Ova-specific CD138⁺/intracellular Ova-binding plasma cells (Ova-PC), and CD61⁺ megakaryocytes (MK) were quantified 3 weeks after secondary immunization by flow cytometry (supplemental Figure 3). Absolute cell numbers were calculated from frequencies determined by flow cytometry and total cell numbers per organ as described in “Calculation of contact frequencies per cell surface area.” Data are mean ± SD for groups of 6 mice. (B) Colocalization frequencies between total plasma cells and CD41⁺ megakaryocytes (MK), B220⁺ (BC), or Gr-1⁺ (Gran) cells at days 6, 9, and 130 after secondary immunization. The environments of 150 to 200 plasma cells were analyzed at each time point. B220⁺ and Gr-1⁺ cells represent approximately 20% and 30% of total bone marrow cells, whereas megakaryocytes represent only approximately 0.3% of cells in this tissue. (C) Relative contacts between plasma cells and B cells (BC), granulocytes (Gran), or megakaryocytes (MK) normalized on the basis of the surface areas of the respective whole populations as calculated in “Calculation of contact frequencies per cell surface area.” (D) Colocalization frequencies between megakaryocytes and IgA, IgG1, or IgM expressing plasma cells as detected in unimmunized mice. (E) Bone marrow (femur) tissue section stained for CD41 (green), κ light chain (red), and f-actin (blue). (F) F-actin density was quantified along the plasma cell megakaryocyte contact areas using Leica Confocal software (LCS Lite Version 2.61 Build 1538). Histograms show intensity of Ig κ and f-actin staining along the 4 lines within and adjacent to a megakaryocyte indicated on the bone marrow section in corresponding colors (left). The green and brown lines cross the plasma cell megakaryocyte contact areas. Here, f-actin and Ig κ intensity reached a maximum. (Top histograms) Ig kappa. (Bottom histogram) f-actin.