Delineation of surface vs intracellular FcεRI/surface MHC II+ cells in three-week old BMMC. For viable cell staining, 1 × 106 three-week old mast cells were stained with FITC-anti-FcεRIα (TW) and PE-anti-IAd at 10 μg/ml and fixed with 1% paraformaldehyde. Panel A: FcεRI + cells were divided into FcεRIlo (below 0.8 to the second log TW intensity), and FcεRIhi cells (between second and third log TW intensity). Accordingly, quadrant #2 (.6%), #3 (35%) and #4b (13.5%) amounted to 49.1% FcεRIhi cells. By contrast, quadrant 1a (9.8%) and #1b (41%) amounted to 49.8% FcεRIlo cells. In total, ~99% cells were FcεRI + cells. Panel B: To determine intracellular distribution, cells were prefixed, permeabilized, and stained. Quadrant percentages were: quadrant #1 (.5%), #2 (12%), #3 (87%), and #4 (0.5%). Approximately 99% cells were FcεRIhi mast cells, and 87% of these mast cells also exhibited high levels of intracellular MHC II.