In Vivo Actin Polymerization Assays.
1.5 × 106 thymocytes obtained from WAS−/ −ΔGBD mice or by transfection of WAS−/ − thymic cells with the pEGFP-C3 WASp Y→F mutant constructs were either unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for 30 min on ice followed by cross-linking with 5 μg mouse anti–hamster IgG for 5 min at 37°C. Activation was terminated and the cells were fixed in 5% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min on ice, washed and stained with 5 μg/ml FITC-conjugated phalloidin, and analyzed using a FACSCalibur™.