Figure 3. Non-specific A2/K^b tetramer binding is influenced by CD8 cell surface density.

A and B. 2×10⁵ 293T cells were incubated +/− 10 μg/ml of the PE-conjugated tetramers A2 D227K/T228A ILAKFLHWL, A2 ILAKFLHWL or A2/K^b ILAKFLHWL for 20 minutes at 37°C, then stained with 7-AAD and either FITC-conjugated anti-CD8 or PE-conjugated anti-CD8β for 30 minutes on ice, washed twice and resuspended in PBS. C. 2.5×10⁵ PBMC were stained with PerCP-conjugated anti-CD8, 7-AAD and either FITC-conjugated anti-αβ-TCR, APC-conjugated anti-CD56 or PE-conjugated anti-γδ-TCR for 30 minutes on ice, washed twice and resuspended in PBS. In A, B and C, data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software.