Figure 4: B-cell differentiation is decreased by the inhibition of NFAT signaling in osteoblasts. (a-b) Bone marrow was flushed from tibiae and femora of 12-week-old control and dnNFATOB mice and analyzed by flow cytometry to examine B-cell development. Percentages of B-lineage cells were determined for control () and dnNFATOB () mice from the gated lymphocyte population. (a) B220+CD19-IgM- (b) B220+CD19+IgM+. Mean indicated by black bar. (c-d) Primary osteoblasts were harvested from calvariae of 1-day-old control and dnNFATOB mice and cultured for 14 days in the presence of -glycerophosphate and ascorbic acid-2-phosphate to induce osteoblast differentiation. Bone marrow was flushed from 12-week-old control mice, and LSK cells were sorted by flow cytometry, seeded on differentiated primary osteoblasts, and cocultured for 14 days. At the end of the culture, osteoblasts and hematopoietic cells were trypsinized and analyzed by flow cytometry for the B220 cell marker. (c) Representative phase-contrast images show hematopoietic cells (indicated by red pseudocolor) cocultured on primary osteoblasts. (d) B220+ B-lineage cells were analyzed by flow cytometry after coculture of LSK cells on control and dnNFATOB differentiated primary osteoblasts. Values, obtained from three independent experiments performed in duplicate, represent the mean ± SD; * compared to control.