Fig. 4.

Dasatinib enhances the visualization of antigen-specific CD8⁺ T-cells in mixed cell populations. A. Staining of HLA A2-restricted CTL lines expanded from PBMC by one round of stimulation with the influenza matrix M1_58-66 peptide (GILGFVFTL) or the Melan-A/Mart-1_26-35 peptide (ELAGIGILTV). Lines were stained with cognate tetramer ± pre-treatment with 50 nM dasatinib for 30 min at 37 °C. B. Flow cytometric profiles of live CD3⁺ lymphocytes stained with HLA A2 tetramers folded around the EBV BMLF1_259-267 (GLCTLVAML), CMV pp65_495-503 (NLVPMVATV) or Melan-A/Mart-1_26-35 (ELAGIGILTV) peptide epitopes. 2x10⁶ PBMC were stained with the amine-reactive viability dye ViViD, then stained with tetramer (1μg in minimal staining volume) ± pre-treatment with dasatinib for 30 min at 37 °C. Cells were then stained with cell surface markers as described in the Materials and methods; a dump channel was used to exclude dead cells, CD14⁺ and CD19⁺ cells from the analysis. Boolean gating was carried out to exclude aggregates. Data were acquired with a BD LSR II flow cytometer and analyzed using FlowJo software.