Serial dilutions of aTc, atovaquone, quinine, and bafilomycin A were generated to yield final concentrations ranging from 40–0.462 nM, 25–0.048 nM, 432–0.84 nM, and 40–0.08 nM, respectively. Synchronous ring-stage PfRAP01 and PfRAP21 conditional knockdown lines as well as a control cell line expressing an aptamer-regulatable fluorescent protein were maintained in high aTc (500 nM), low aTc in the case of PfRAP01 (8 nM) and PfRAP21 (5 nM) or no aTc, and were distributed into 384-well assay plates (Corning®, 89176-442). Compounds were transferred to the parasite-containing plates using the Janus® platform (PerkinElmer). DMSO- and dihydroartemisinin-treatment (500 nM) served as reference controls. Growth inhibition was analyzed after 72 and 120 h using the Renilla-Glo(R) Luciferase Assay System (Promega, E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). IC50 values were obtained from corrected dose-response curves and plotted using GraphPad Prism 8.
Renilla glo r luciferase assay system
Manufactured by Promega
The Renilla-Glo(R) Luciferase Assay System is a laboratory equipment used for the detection and quantification of Renilla luciferase activity. It provides a bioluminescent assay for sensitive measurement of Renilla luciferase expression in cell-based systems.
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12 protocols using renilla glo r luciferase assay system
1
Conditional Parasite Growth Inhibition Assay
2
Luminescence-based Parasite Growth Assay
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Assessment of parasite proliferation rate in the presence and absence of aTc was carried out using luminescence as a readout of growth. In 96-well U-bottom BD Falcon™ plates, synchronous ring-stage parasites were set up in triplicate and cultured in the presence (0.5 μM) and absence of aTc and luminescence measured at 0 and 72 h by quantitating luminescence using the Renilla-Glo(R) Luciferase Assay System (Promega E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). Parasite growth was determined from normalized RLuc values, with dihydroartemisinin- (DHA) treated (0.5 μM) samples as a no growth control. Data were analyzed using GraphPad Prism Version 8.
3
Quantifying Pf Parasite Growth Dynamics
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To assess the growth of PfAcAS conditional knockdown parasites, synchronous ring-stage parasites were cultured in the presence (50 and 3 nM) and absence of aTc and set up in triplicate in a 96-well U-bottom plate (Corning 62,406-121). Luminescence was measured at 0, 72, and 120 hr post-invasion using the Renilla-Glo(R) Luciferase Assay System (Promega E2750) and the GloMax Discover Multimode Microplate Reader (Promega). The luminescence values were normalized to chloroquine-treated (200 nM) samples and results were visualized on a scatterplot using GraphPad Prism (version 8; GraphPad Software).
4
Parasite Proliferation Kinetics Assay
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Parasite proliferation rates were assessed in the presence and absence of aTc, using luminescence as a readout of growth. Synchronous ring-stage parasites were set up in triplicate in 96-well U-bottom BD Falcon™ plates and cultured in the presence (50 nM) and absence of aTc. Expansion was measured at 0 and 72 h by quantifying luminescence using the Renilla-Glo(R) Luciferase Assay System (Promega E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). Parasite growth was determined from normalized RLuc values, with samples treated with 200 nM chloroquine (a lethal concentration) included as no-growth controls. Results were processed and visualized using Prism 8 (GraphPad Software). Experiments were performed on two independent occasions and statistical significance was assessed using an unpaired t test.
5
Quantifying Parasite Growth Dynamics
6
Investigating PfACG1 and PfEHD Perturbation
7
Measuring Plasmodium Proliferation by ACS10
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Assessment of parasite proliferation rate over two intra-erythrocytic developmental cycles by titrating the expression of PfACS10 and YFP was carried out by maintaining the cultures in varying aTc concentrations and using luminescence as a readout of growth. In a 96-well U-bottom BD Falcon™ plate, synchronous ring-stage parasites were set up in triplicate and cultured in the presence (50 and 3 nM) or absence of aTc. Expansion was measured at 0, 72, and 120 h by quantitating luminescence using the Renilla-Glo(R) Luciferase Assay System (ref no E2750, Promega) and the GloMax® Discover Multimode Microplate Reader (Promega). The luminescence values were normalized to chloroquine-treated (200 nM) samples, and results were visualized on a scatter plot using GraphPad Prism (version 8; GraphPad Software).
8
Measuring Parasite Viability Dynamics
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Assessment of parasite viability during target protein perturbations was carried out using luminescence as a readout of growth. Synchronous ring-stage parasites, cultured in the presence (50 nM) and absence of aTc, were set up in triplicate in a 96-well U-bottom plates (Corning® 62406-121). Luminescence signals were taken at 0 and 72 h post-invasion using the Renilla-Glo(R) Luciferase Assay System (Promega E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). The luminescence values in the knockdown conditions were normalized to aTc-treated (100% growth) and dihydroartemisinin-treated (500 nM, no growth) samples and results were visualized using GraphPad Prism (version 9; GraphPad Software).
9
Parasite Growth Kinetics in KD Lines
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Assessment of parasite proliferation rates in β2 and β5 cKD lines upon knockdown of protein expression in varying aTc concentrations was carried out using luminescence as a readout for growth. Synchronous ring-stage parasites cultured in 0 nM aTc or increasing aTc concentrations (15, 20 or 500 nM for β2, and 10, 20 or 500 nM for β5) were set up in triplicate in 96-well plates and luminescence signals were taken at 0, 24, and 72 h post-invasion using the Renilla-Glo(R) Luciferase Assay System (Promega E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). Results were visualized using GraphPad Prism.
10
Parasite Proliferation Dynamics in β2 and β5 cKD
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