pCD19 recruitment to BCR aggregation is reduced in Mst1 KO B cells after stimulation. Splenic B cells from WT and Mst1 KO mice were incubated with AF546–mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained for pCD19 using a specific monoclonal antibody and AF488-conjugated secondary antibody. Cells were analyzed using TIRFm (A-B). The MFI of pCD19 staining in the B-cell contact zone was quantified (C). Shown are representative images and TIRFm analysis of the spatial relationship of BCR with pCD19 in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-Ig. The correlation coefficients between BCR and pCD19 staining were determined using NIS-Elements AR 3.2 software (D). Splenic B cells were incubated with AF546–mB-Fab′–anti-Ig without (0 minutes) or with sAg at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for pCD19 and analyzed using CFm (E-F). The MIF of pCD19 was generated using NIS-Elements AR 3.2 software (G). The Pearson’s correlation coefficients between BCR and pCD19 staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software (H). Shown are representative images at indicated times and the average values (±SD) of ∼50 cells from 3 independent experiments. Scale bars, 2.5 μm. *P < .01.