Agonist-induced calcium response in single human platelets assayed in a micro fluidic device
Louie Tran, Javier Farinas, Lily Ruslim-Litrus, Pamela B. Conley, Craig Muir, Kevin Munnelly, David M. Sedlock, Diana B. CherbavazTo 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 micro fluidics-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 micro-fluidic chip-based assay. For the micro uidic device used in these studies, a cell density of 1 to 2x106 platelets per milliliter and a nominal ow 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 micro fluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.
March 14, 2005
Analytical Biochemistry
Year: 2005. Volume: 341.
