3.2. Optimization of SMM Screening Using Living Cells
In order to optimize screening parameters of our SMM using living cells, cRGDyK spots were arrayed, and a mixture of M21 and M21-L cells were applied while systematically varying motion, incubation time, ligand spotting concentration, and number of panned cells (Figure 2A). Notably, cell binding was greatly improved in the absence of motion, a result that might be explained by a boundary layer of shear stress created immediately above the surface of the slide in the presence of motion. Once stationary, cell binding increased linearly with incubation time up to 120 min, at which time saturation occurred. Cell binding also increased as a function ligand spotting concentration, with saturation occurring above 0.25 mM. The number of panned cells also increased binding, with saturation occurring at 4 × 106 cells per slide. To maximize the specificity of binding, we compared incubation in the presence or absence of 10% serum, and with or without negative control cells (Figure 2B). Both serum and competing cells improved specificity, likely by blocking non-specific interactions.
Figure 2  Optimization of screening parameters: (A) Maximizing sensitivity through the effect of motion, incubation time, ligand spotting concentration, and the number of panned cells using a cRGDyK array and a mixture of M21 and M21-L cells. Shown are mean ± SD for each data point from 4 randomly chosen spots on the slide. (B) Maximizing specificity through the use of serum or competing receptor-negative cells.

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