c-kit⁺ FcεRI⁺ cells derived from Ik^−/− bone marrow cultured under mast cell differentiation conditions exhibit developmental abnormalities. Ik^+/+ and Ik^−/− bone marrow was cultured in IL-3 and SCF for 6 weeks. (A) Analysis of c-kit and FcεRI expression by flow cytometry. (B) Ik^+/+ and Ik^−/− cultures stained with Wright-Giemsa (original magnification ×100). (C) β-hexosaminidase release was determined 30 minutes after FcεRI crosslinking of pre-loaded anti–DNP-IgE and indicated concentrations of DNP-human serum albumin. The percentage of β-hexosaminidase release was calculated as release (%) = supernatant/(supernatant + cell lysate) × 100. Data represents mean of triplicate experiments ± standard error of the mean. ***P < .001 in 10 ng/mL DNP-human serum albumin-treated cultures. (D) Cytokine release at 16 hours postactivation with anti–DNP-IgE and DNP (10 ng/mL) determined by enzyme-linked immunosorbent assay. Data represents mean of replicates from 3 independent cultures ± standard error of the mean. (E) Basal expression of cell-surface adhesion and activation molecules after 6 weeks in culture. (F) Quantitative reverse transcription PCR analysis of mast cell and basophil lineage-specifying genes in Ik^+/+ and Ik^−/− cultures. Data are expressed as the mean of 3 separate quantitative RT-PCR analyses ± standard deviation using RNA isolated from same bone marrow culture. *P < .05. Results in panels A-E are representative of 3 independent experiments, and results in panel F are representative of 2 independent experiments.