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®) was kindly provided by the Walter Reed Army Institute of Research. Currently the throughput of this assay is approximately 100 compounds per day. Recently we have added a High Throughput Sampler to our existing LSR2 from Becton-Dickinson allowing for 300-900 compounds (or drug dilutions) to be tested per day. The rate limiting step is dilution of samples to the appropriate concentration.
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50 values using GraphPad Prism version 5.00 for Windows (San Diego,CA).In the future, collaborations with Verity Software House will allow the authors access to Gemstone automated FACS analysis software (Beta version) to deal with the vast amounts of data generated with our high content analysis. FACS plots can be found in Grimberg et al. (2008) (Ref. 7).We would like to emphasize to the malarial research community and to those not familiar with flow cytometry techniques that the essence of this Letter describes the first application of this new flow cytometry method to measure the stage in vitro drug susceptibility of the 3 known antimalarials and to discover new antimalarials. Additional details as to how the flow cytometry method enables these measurements and analysis and its preliminary validation and optimization in comparison to hypoxanthine uptake measurements can be found in Ref. 7.
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The data generated for compound #13 demonstrate an order of magnitude less growth inhibition for the trophozoite stage when compared to the ring and schizont stages. This may suggest that this compound is preventing maturation of the trophozoite stage to the schizont stage. Presently, we do not have additional supporting data. However, it points to the fact that our methodology even with the use of asynchronous cultures enables the detection of compounds that have unique stage growth inhibition profiles.
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Cell size is indicated by the forward scatter (FSC) on a flow cytometer. This parameter is proportional to the amount of laser light passing unobstructed around a cell.
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Internal complexity is indicated by the side scatter (SSC) on a flow cytometer. This parameter is proportional to the amount of laser light which is reflected by cell membranes and organelles.
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In addition to changes in cell shape, drug toxicity to cells can be detected as damage to the membrane and is indicated by the presence of propidium iodide stain within a cell.
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