Characterization of the BMMC and macrophages used for transfer experiment. (A) Surface expression of FcɛR, c-Kit, FcγRII/III, Mac-1, and Gr-1 on BMMC. BMMC from FcRγ +/+, FcγR−/−, and TNFβ−/− mice were stained with biotinylated IgE plus avidin-PE, anti–c-Kit, 2.4G2, anti–Mac-1 MoAb, and anti–Gr-1 MoAb. The dotted line indicates control staining. (B) β-hexosaminidase assay of BMMC. BMMC from FcRγ +/+, FcγR−/−, and TNFβ−/− mice were stimulated by FcɛRI cross-linking by biotin-IgE and avidin or by Ca²⁺-ionophore for 30 minutes. β-hexosaminidase activity in the supernatants and the lysate of the cell pellets was quantified by spectrophotometric analysis as described in Materials and Methods. Total β-hexosaminidase content (supernatant activity plus lysate activity) was similar in these cells (data not shown). (C) FcγR-induced release of TNF. Macrophages and BMMC from FcRγ +/+, FcRγ−/−, and TNFβ−/− mice were stimulated with or without 50 μg/mL immobilized IgG1 or 12 μg/mL immobilized 2.4G2 for 30 minutes. TNF released into the media was measured by ELISA.