Phenotypic and functional characterization of PCMCs. (A) CD117 and FcϵRI expression on PCMC surface. The number in the top right indicates the percentage of double-positive cells. (B) Mast cell tryptase staining by indirect immunofluorescence with an Alexa 633–conjugated secondary Ab (blue). Samples were inspected with a confocal laser-scanning microscope. (C-D) Histochemical staining of PCMCs with toluidine blue (C) or Alcian blue followed by safranin (D). Bars = 10 μm. (E) Secretagogue-induced β-hexosaminidase release. PCMCs were stimulated with increasing concentrations of Substance P or compound 48/80 for 30 minutes at 37°C. (F) FcϵRI-dependent β-hexosaminidase release. PCMCs were sensitized with mouse IgE anti-DNP and challenged with DNP-HSA. The percentage of β-hexosaminidase released was plotted against the concentration of the stimulus. Tests for phenotypic and functional analysis of PCMCs were routinely performed after 3 to 5 weeks of culture. Data are from 1 representative experiment of 3.