3.1. Live Cell Imaging and Controls
To validate the assay, integrin αvβ3-positive M21 cells (positive control) labeled with the 700 nm NIR fluorophore ESNF10 and integrin αvβ3-negative M21-L cells (negative control) labeled with the 800 nm NIR fluorophore IR786 were panned over the surface of our SMM (Figure 1A). PAAm, a “sticky” cationic polymer showing no specificity to cell surfaces was used as a positive ligand control, which bound all cell types. Using dual-channel NIR fluorescence microscopy, the number of individual cells binding each spot could be counted (Figure 1B). Thus the readout of our assay was number of cells bound per spot, with the theoretical maximum number of bound cells (i.e., the dynamic range of the assay) being defined by the PAAm control spots (≈300 cells per spot for all cell lines tested).
Results of the assay using the integrin-binding peptide cRGDyK as the ligand spot are shown in Figure 1B. Specificity was defined in one of two ways. In the absence of negative control cells, specificity was the number of receptor-positive cells binding a ligand spot divided by the number of these same cells binding inter-spot blank space on the slide. In the presence of negative control cells, specificity was the number of receptor-positive cells binding a ligand spot minus the number of receptor-negative cells bound to that same spot. Sensitivity was defined as the absolute number of receptor-positive cells bound to a particular spot. Of note, pseudo-coloring of 700 nm fluorescence in red and the 800 nm fluorescence in green permitted rapid visual assessment of specificity as demonstrated in Figure 1.
Figure 1  Dual-channel screening strategy and controls. (A) Living integrin αvβ3-positive M21 cells (target cells; stained with ESNF10 and pseudo-colored in red) and integrin αvβ3‑negative M21-L cells (control cells; stained with IR786 and pseudo-colored in green) prior to dissociation from their respective plates. Scale bars = 100 μm. (B) The same cells mixed together and panned over PAAm positive control spots (top row) or cRGDyK ligand spots (bottom row). The yellow color indicates co-localized M21 and M21-L cells. Scale bars = 300 μm.

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