Aptamer and Ab binding assay

A549, HeLa, H1975, H820, and MCF7 cells were seeded at 40,000–50,000 cells/well in a 48-well flat-bottom tissue culture plate 24 h before experiment. On the day of the assay, aptamers were folded and annealed as described above. APC-labeled EGFR and c-Met mAbs and isotype control were diluted to desired concentration in DPBS supplemented with 0.1% BSA. Cells were washed once with DPBS and then incubated in either aptamer or Ab binding solution for 45 min at 37°C and 5% CO₂. After incubation, the solution was removed, cells were washed once with DPBS, and cells were lifted off with 1× TrypLE Express (Thermo Fisher, Waltham, MA) for <5 min at 37°C. TrypLE Express was diluted by the addition of complete medium, and cells were transferred to 1.5-mL Eppendorf tubes and gently centrifuged to pellet (5 min, 400 × g). Cells were then fixed in 4% paraformaldehyde (PFA) in DPBS in the dark for 5 min at 4°C before being pelleted (5 min, 400 × g) and resuspended in DPBS. Cells were kept in the dark at 4°C until analysis with flow cytometry. Transiently transfected 293FT cells were subjected to the same procedure but were not washed at either step due to their poor adherence to the plates.