Impaired maturation of Btk-deficient bone marrow derived neutrophils. (A) The maturation status of granulocytes in the bone marrow can be distinguished by the expression of CD11b and Gr-1. To prove the settings, gated cells were sorted and Pappenheim-staining on cytospins were performed. (B) Mice were treated with BrdU for 4 days and the Gr-1⁺ subpopulations, according to panel A, were analyzed for BrdU incorporation. (C) The granulocyte maturation phenotype of wild-type (wt) or Btk-deficient (Btk-ko) mice was analyzed on freshly isolated and erythrocytes-depleted bone marrow cells or (D) on for 20 hours in the presence of GM-CSF-matured bone marrow-derived neutrophils. The degree of maturation was defined as depicted in panel A. The percentage of immature and mature neutrophils relative to the overall CD11b⁺/Gr-1⁺ population is shown. (E) Wild-type and Btk-deficient bone marrow-derived granulocytes were analyzed by electron microscopy. Granules were counted per cell and per μm² cytosol using ImageJ software. (F) The expression of granule proteins was analyzed by quantitative PCR (Elane = neutrophils elastase, Ltf = lactotransferrin precursor, Mpo = myeloperoxidase) relative to the expression of β-actin (Actb). The release of elastase (G) or gelatinase (H) upon IC-induced degranulation of wild-type or Btk-deficient granulocytes is presented. Data presented are the mean values (± SD). **P ≤ 0.005; ***P ≤ 0.0005. n represents the number of biological replicates.