High-throughput growth assay for XXXX using yellow fluorescent protein.

@entity3442 @entity11119 @entity4765 @entity204 @entity3402 @entity3204
A high-throughput growth assay for the protozoan parasite @entity3204 was developed based on a highly fluorescent transgenic parasite line. These parasites are stably transfected with a tandem yellow fluorescent protein (YFP) and are 1,000 times more fluorescent than the wild type. Parasites were inoculated in optical-bottom 384-well culture plates containing a confluent monolayer of host cells, and growth was monitored by using a fluorescence plate reader. The signal was linearly correlated with parasite numbers over a wide array. Direct comparison of the YFP growth assay with the beta-galactosidase growth assay by using parasites expressing both reporters demonstrated that the assays' sensitivities were comparable but that the accuracy of the YFP assay was higher, especially at higher numbers of parasites per well. Determination of the 50%-inhibitory concentrations of three known growth-inhibiting drugs ( @entity11119 , @entity4765 , and @entity3402 ) resulted in values comparable to published data. The delayed parasite @entity204 kinetics of @entity3402 could be measured without modification of the assay, making this assay very versatile. Additionally, the temperature-dependent effect of @entity4765 was assayed in both wild-type and engineered drug-resistant parasites. Lastly, the development of @entity3442 resistance after transfection of a resistance gene in @entity3204 was followed. In conclusion, the YFP growth assay limits pipetting steps to a minimum, is highly versatile and amendable to automation, and should enable rapid screening of compounds to fulfill the need for more efficient and less toxic antiparasitic drugs.
