A stock solution of sapanisertib was serially diluted to yield final concentrations ranging from 1 to 500 nM in the assay. Synchronous ring-stage PKG (PF3D7_1436600) (12 (link)), PI4Kβ (PF3D7_0509800), and PI3K (PF3D7_0515300) cKD parasite lines, as well as a control cell line expressing an aptamer-regulatable fluorescent protein, were maintained in the presence of high aTc (500 nM) or low aTc (2 nM in the case of PKG and no aTc in the case of PI4Kβ and PI3K) and distributed into 384-well polystyrene microplates (Corning). Compounds were transferred to the parasite-containing plates using the Janus platform (PerkinElmer). DMSO and dihydroartemisinin treatment (500 nM) served as reference controls. Luminescence was measured after 72 hours using the Renilla-Glo Luciferase Assay System (Promega E2750) and the GloMax Discover Multimode Microplate Reader (Promega), and IC50 values were obtained from corrected dose-response curves using Graph-Pad Prism.
384 well polystyrene microplates
Manufactured by Corning
384-well polystyrene microplates are a type of lab equipment used for various applications in scientific research and experiments. These microplates feature a grid of 384 individual wells, each designed to hold a small volume of liquid samples. The plates are made of polystyrene, a common and durable plastic material. The primary function of these microplates is to provide a standardized and efficient platform for performing tasks such as sample preparation, assays, and high-throughput screening.
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2 protocols using 384 well polystyrene microplates
1
Sapanisertib Dose-Response Assay in Malaria Parasites
2
Antimalarial Compounds Dose-Response Assay
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Stock solutions of compounds were serially diluted to yield final in-assay concentrations ranging from 0.005-2.8 μM for harzianin NPDG H and alamethicin, 0.0006-0.312 μM for harzianin NPDG I, 0.008-4 μM for trichorzin HA II, and 0.05-0.00009 μM for mefloquine (positive control). As a control cell line, a parasite line expressing an aptamer-regulatable fluorescent protein integrated in the cg6 locus84 (link) was assayed in parallel. Synchronous ring-stage parasites in the presence (0.5 μM) and absence of aTc were dispensed into 384-well polystyrene microplates (Corning®), and compounds added using the Janus® platform (PerkinElmer). DMSO- and DHA (0.5 μM) served as normalization controls for maximal and minimum signal. Luminescence was measured after 72 h and IC50 values were obtained from normalized dose-response curves using GraphPad Prism Version 8.
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