Flow cytometry has traditionally been used for the analysis of individual samples. Our invention, HyperCyt uses a peristaltic pump in combination with an autosampler to boost throughput (see References 1- 13 in Center Overview). As the sampling probe moves from well to well, a peristaltic pump sequentially aspirates particle suspensions from each well. Between wells, the pump draws a bubble of air into the sample line resulting in the delivery of a series of bubble-separated samples. The time-resolved data, with periodic gaps corresponding to the passage of the sample-separating air bubbles, are analyzed by our software. When the MLI began, we had screened only 96 well plates, but proposed to move to a fully automated 384 well system. We routinely screen 384 well plates with multiplexed data sets in 10-12 min with sample volumes of 1- 2 u,l, even for multiplexed samples. Complete systems integration has proven elusive in part because flow cytometry companies had been reluctant, except for Luminex which provided an SDK, to provide access to proprietary control software which allows integration of our sampling system with the flow cytometer. (Although we have shown that Luminex 20 plexes are compatible with NTS, the cost of Luminex reagents ~$0.10 per well per singleplex even at a deep bulk discount would add $250,000 to the cost of screening a sixplex against a library of 500,000 compounds.) We have recently solved this generic control problem by creating our own software that controls third party flow cytometry programs via the Windows API (application programming interface) and by collaborating with LANL to implement a free standing data acquisition system, ORCAS. We are now in a position to use our expertise and tools to develop a fully integrated HT flow cytometry system and to overcome the limitations of flow cytometry to complement fully automated technologies in the MLPCN. We will: 1) overcome the throughput barrier by rapid sampling from 1536 well plates;2) overcome the fluid carryover limitation that arises from sampling through long tubing via a peristaltic pump in front of the sample chamber;3) overcome the limitation of cell compression and activation by the peristaltic pump;4) overcome the limitation of dilute samples when adherent cell populations are resuspended in 384 or 1536 well plates. By the end of 4 years, we will integrate these elements into several, robust automated platforms that include sample plate thermal control and particle resuspension, allowing NTS flow cytometry to take its place in the armamentarium of robust HTS capabilities for the MLPCN and for the international scientific community.
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