Abstract
The screening of living cells using high-throughput microarrays is technically challenging. Great care must be taken in the chemical presentation of potential ligands and the number of collisions that cells make with them. To overcome these issues, we have developed a glass slide-based microarray system to discover small molecule ligands that preferentially bind to one cell type over another, including when the cells differ by only a single receptor. Chemical spots of 300 ± 10 μm in diameter are conjugated covalently to glass slides using an arraying robot, and novel near-infrared fluorophores with peak emission at 700 nm and 800 nm are used to label two different cell types. By carefully optimizing incubation conditions, including cell density, motion, kinetics, detection, etc. we demonstrate that cell-ligand binding occurs, and that the number of cells bound per chemical spot correlates with ligand affinity and specificity. This screening system lays the foundation for high-throughput discovery of novel ligands to the cell surface.
Highlights
Extracellular membrane (ECM) receptors are of significant importance to the development of new therapeutic agents, being the molecular targets for more than 60% of clinical drugs [1,2]
We have previously described functionalized microarray slides capable of rapid and high-throughput screening of over 5000 different chemical compounds binding to living bacteria, including quantitation of binding parameters [19,20,21]
Integrin αvβ3-positive M21 cells labeled with the 700 nm NIR fluorophore ESNF10 and integrin αvβ3-negative M21-L cells labeled with the 800 nm NIR fluorophore IR786 were panned over the surface of our small molecule microarray (SMM) (Figure 1A)
Summary
Extracellular membrane (ECM) receptors are of significant importance to the development of new therapeutic agents, being the molecular targets for more than 60% of clinical drugs [1,2]. High-throughput SMM screening is currently limited due to the use of non-physiological contexts (e.g., absence of serum) and non-viable samples (e.g., cell lysates); most current methods neglect ECM dynamics [12,13,14]. We optimized the key experimental parameters for screening living mammalian cells using known small molecule ligands on the previously developed SMM, which requires simultaneous optimization of ligand presentation, the effect of motion, incubation time, ligand concentration, and the number of panned cells
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