Figure 3.

(A) Total immunofluoresence labeling of RBL-2H3 cells with CT2 antibodies does not change after antigen stimulation. Suspension cultures of DNP-specific, IgE-primed RBL-2H3 cells were fixed before (dotted and solid lines) or after (dashed line) 10-min activation with antigen (1 μg/ml DNP-BSA). Permeabilized cells were stained with CT2 antibodies directed at type II InsP₃ receptors (solid and dashed lines), followed by FITC-conjugated secondary antibodies, or with secondary antibody alone (dotted line). Fluorescence was quantitated by flow cytometry. (B) Receptor aggregates increase after antigen stimulation. Coverslips were prepared as described in the legend to Figure 2 and documented using a Zeiss Photomicroscope III equipped with a computer-interfaced Photometrics charge-coupled device camera. Images were acquired for a minimum of 15 fields, totaling >70 cells for each condition. Image analysis was performed using Compix Simple software. Cell boundaries were manually defined in each field, and a threshold was set for object recognition using the Sobel function. Data are expressed as objects per unit area ± SEM; significance was determined using the unpaired t test (p < 0.0001).