Cell-based screening: a high throughput flow cytometry platform for identification of cell-specific targeting molecules.
Targeting of drugs and genes to specific cell types is an emerging paradigm in the treatment of many medical conditions. However, targeting structures such as peptides are susceptible to rapid inactivation in vivo. To address this problem, novel targeting molecules can now be rapidly synthesized using a combinatorial approach. Methods to screen the large libraries created in this process are often lacking or compatible only with solution-based screening. This report describes a high-throughput cell-based method utilizing flow cytometry, capable of rapidly screening large libraries of molecules simultaneously for biological functionality and stability. In this method, each library molecule is attached to a microsphere exhibiting a unique set of optical properties, or "fingerprint", conferring modularity and multiplex capability. We investigated the multiplex capability of our flow cytometric method to determine its capacity for high-throughput screening. Current instrumentation in our laboratory allows the screening of at least 75 unique compounds in a single well, a number comparable to available solution-based assays. In state-of-the-art configuration, however, this methodology can support the screening of up to 1875 compounds per well, achieving high-throughput potential in a single multiwell plate. We also investigated the binding capability of targeted microspheres to adherent target cells. These microspheres exhibited a 12-fold increase in binding over control, untargeted microspheres. Competitive inhibition experiments with soluble ligand confirmed the specificity of microsphere binding. Overall, the methodology proposed here is capable of quickly and effectively screening large libraries of targeting molecules using instrumentation readily available to the greater research community.
Smith
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Vanderbilt University, Box 351620, Station B, Nashville, TN 37235, USA.
Giorgio
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D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D008863:Microspheres; D011956:Receptors, Cell Surface / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface; D001710:Biotin