NOS activity and nitric oxide localization in human mast cells. (A) NOS activity in HMC-1 cells, as measured using the [¹⁴C]-l-arginine conversion assay. Brain homogenate was used as a positive control. Each sample was run in the presence of EGTA (2 mM) to determine the levels of calcium-dependent (constitutive) NOS activity. The results are expressed as picomoles l-citrulline formed per minute, per milligram of protein. Data shown as mean ± SEM for 3 independent experiments. (B) Measurement of endogenous NO formation in HMC-1 and LAD 2 cells as detected by DAF fluorescence. Cells were loaded with DAF, basal fluorescence images were obtained, and then HMC-1 cells were stimulated with A23187 (0.1 μM) or LAD 2 cells stimulated by IgE cross-linking (1 μg/mL). Changes in fluorescence were captured using confocal microscopy 10 minutes after activation. Most HMC-1 cells (75% ± 6.3%; n = 4) were positive, whereas fewer LAD 2 cells (42% ± 1.6%, n = 3) were positive. White arrowheads show nuclear/perinuclear DAF fluorescence. White arrows indicate DAF-negative cells that also show cytoplasmic ruffling (HMC-1 and LAD 2) or granule release (LAD 2). Original magnification × 800; bar = 10 μm. Results are representative of at least 3 independent experiments.