Agonist-induced calcium response in single human platelets assayed in a microfluidic device |
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Authors: | Tran Louie Farinas Javier Ruslim-Litrus Lily Conley Pamela B Muir Craig Munnelly Kevin Sedlock David M Cherbavaz Diana B |
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Affiliation: | Caliper Technologies, Mountain View, CA 94043, USA. |
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Abstract: | To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets. |
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Keywords: | ADP Assay Development Calcium flux Fura-2 High-throughput screening Indo-1 Microfluidic chip Microfluidics Nanoliter Ratiometric fluorometry PAR-1 TRAP |
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