The recruitment of Mst1 to BCR aggregates in B cells stimulated by sAg or mAg. To mimic sAg, splenic B cells were incubated with AF546–mB-Fab′–anti-Ig for 10 minutes at 4°C to label the BCR. Then, the cells were incubated with either streptavidin or the medium alone (0 minutes) as a control at 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for pMst1 and analyzed using CFm (A). Images were quantitatively analyzed to determine the fluorescence intensity of cell-associated pMst1 (C) and the correlation coefficients between the labeled BCR and pMst1 (E). To mimic mAg, splenic B cells were incubated with AF546–mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for varying lengths of time. As controls, splenic B cells were labeled with AF546–Fab–anti-Ig for the BCR before incubation with biotinylated transferrin-tethered lipid bilayers. After fixation and permeabilization, the cells were stained for pMst1 and analyzed using TIRFm (B). The MFI of pMst1 (D) in the B-cell contact zone and the correlation coefficients (F) between the BCR and pMst1 were quantified using TIRFm images and NIS-Elements AR 3.2 software. Shown are representative images and mean values (± standard deviation [SD]) from 3 independent experiments where over 50 cells were individually analyzed using NIS-Elements AR 3.2 software. Scale bars, 2.5 μm. *P < .01. IRM, interference reflection microscopy; Tf, transferrin.