IFA causes reorganization of the bone marrow B-cell environment. (A) The number of sinusoidal B cells and granulocytes was determined by in vivo labeling. Five hundred nanograms of αB220-PE or αLy6G-PE was injected IV 5 minutes prior to euthanizing the mice. Representative flow panels display sinusoidal frequency within gated IgD⁺ or immature (IgD^-IgM⁺CD19⁺). Graphed data are pooled from 3 experiments for B cells or a representative experiment for granulocytes. (B) In vivo sinusoid labeling was performed on splenectomized mice. Shown is a representative experiment. (C) Bone marrow from IFA or untreated Rag1^−/− mice was plated in equal ratios with CD19⁺ naive splenocytes for 18 hours with 10 μg/mL LPS. Data are representative of 3 independent experiments. Error bars are SEM. (D) Cytokine levels in the extracellular fluid of the bone marrow were examined with multiplex ELISA. Data are pooled from 2 experiments. The thresholds of detection are: IL-5, 0.7 pg/mL; IL-6, 1.8 pg/mL; IL-7, 0.9 pg/mL; CCL3, 8.3 pg/mL; IL-13, 6.3; Eotaxin, 4.4 pg/mL. (E) Thy1^loDX5⁺ bone marrow cells were enumerated by flow cytometry. Representative flow plots are shown and graphed data are pooled from 4 experiments. (F) April expression was detected by intracellular flow cytometry. (G) CD11c⁺MHCII^hi, SigLecF⁺Gr-1^loCD11b⁺SSC^hi and CD41⁺SSC^hiFSC^hipolyploid (n > 2) cells were enumerated by flow cytometry. Data are pooled from 2 experiments.