BCR cluster formation, B-cell spreading, and BCR signalsome are reduced in Mst1 KO B cells. 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 pY and pBtk using a specific monoclonal antibody and an AF405-conjugated and AF488-conjugated secondary antibody. Cells were analyzed using TIRFm. Shown are representative images (A-B) and the average values (± SD) of the B-cell contact area (C), the MFI of the BCR (D), and the MFI of the pY (E) and pBtk (F) in the contact zone. TIRFm analysis of the spatial relationship of BCR with pY and pBtk in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-Ig. The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (G). Flow cytometry analysis of the MFI of pY (H) in WT and Mst1 KO B cells without antigen stimulation. The data were generated using 20 to 90 cells from 3 independent experiments. Scale bars, 2.5 μm. *P < .01. NS, not significant.