Comparison of GPVI levels expressed on different clones of transfected RBL-2H3 cells. Surface expression of GPVI was analyzed on 3 different clones of RBL-2H3 cells transfected with human GPVI. (A) GPVI levels were measured on transfected RBL-2H3 cells using mAb 204-11 by quantitative flow cytometry. Cells were stained with mAb 204-11 (10 μg/mL) for 15 minutes, and then an FITC-labeled antimouse antibody was added and incubated for a further 15 minutes in the dark. Simultaneously, calibration beads were incubated with the antimouse FITC secondary for 15 minutes. Diluent was added to all tubes, which were then analyzed by flow cytometry. A calibration curve was constructed and the number of GPVI receptors per cell was determined. (B) GPVI-transfected RBL-2H3 cells were stained with convulxin (10 μg/mL) for 30 minutes, washed, and incubated with a rabbit IgG anticonvulxin antibody for 30 minutes. After further washing, the cells were incubated with anti–rabbit IgG FITC for 30 minutes, then diluted and analyzed by flow cytometry. All results are expressed ± SEM, n = 4.