NFAT Luciferase Reporter Assay.
Murine WAS−/− thymocytes (2 × 107 cells) were isolated and transfected with 50 μg of an NFAT luciferase reporter gene plus 50 μg pEGFP-C3 vectors encoding WASp-GFP, WASp Y→F GFP mutants, or GFP alone. After 4 h, viable cells were separated using Lympholyte M (Cedarlane) and the GFP+ cells were purified over a MoFlo® cell sorter (DakoCytomation) and cultured for 8 h in uncoated 96-well plates or in plates coated with 10 μg/ml anti-CD3 or 5 μg/ml anti-CD3 plus anti-CD28 antibody. Cells were lysed and luciferase activity was assayed using the luciferase assay system (Promega) and a luminometer.