Quantification of Membrane-Bound AKT in Activated CD8 T Cells

Images were taken using Leica AM TIRF MC imaging system as described with the following modifications (Bilal et al., 2015 (link)). P14 CD8 T cells were isolated by positive selection, based on Thy1.1, and placed on glass chamber slides (5 × 10⁴ cells/chamber; LabTek II) precoated with 10 μg/mL α-CD3 mAb. Cells were stimulated for 15 minutes, fixed with 4 % paraformaldehyde, and permeabilized with 0.25 % Triton-X. Cells were blocked with SEA blocking buffer (Thermo-Fisher) for 1 hour and stained with 5 µL rabbit α-human/mouse AKT antibody (11E7, Cell Signaling Technology) overnight at 4 °C. Cells were washed and incubated with DyLight 488-conjugated donkey α-rabbit IgG (poly4064, BioLegend) secondary antibody for 2 hr at room temperature. Cells were washed and fresh PBS was added to each well. Images were taken at room temperature using 100 X oil submersion lens and Leica AM TIRF MC imaging system at the University of Iowa Central Microscopy Research Facility. Laser intensity and exposure parameters remained constant within each experiment. TIRF microscopy images were analyzed using ImageJ software. Membrane AKT was quantified by measuring mean pixel intensity in the longest axis of cells.