Clariostar luminometer

Following the indicated treatment, cells were serum-starved overnight in DMEM supplemented with 5 mM glucose, then incubated for 1 h in glucose-free DMEM +/−100nM insulin. Medium was replaced with PBS + 0.125 mM 2-deoxy glucose (2-DG). Glucose uptake reactions were conducted for 30 min, and then terminated by addition of stop buffer (0.4 M HCl + 2% dodecyl trimethyl ammonium bromide). 2DG6P detection reagent was applied and data were acquired using a CLARIOStar luminometer (BMG Labtech, Ortenberg, Germany).

Sorbitol synchronised Hyp1-Nluc parasites were diluted into a 24-well plate at 4% HCT, 2% ring stage parasitemia with Complete Media. Compounds were added to separate wells in the following concentrations: 1x EC₅₀ M5717 (0.56 nM), 5x EC₅₀ M5717 (2.8 nM), 10x EC₅₀ M5717 (5.6 nM), 5x EC₅₀ artemisinin (40 nM), 5x EC₅₀ chloroquine (64.5 nM), 100 µM cycloheximide and 0.05% DMSO. A blood smear was immediately taken (0 h), fixed in methanol for 30 s and stained in 10% Giemsa for 5 min. An aliquot of resuspended culture was removed at the same time and frozen in a 96-well plate at -20°C. Parasites were returned to 37°C. Over the following 72 h, another blood smear and aliquot were taken at each 24 h time point. Results were determined by counting 1000 cells for each treatment and time point, separating parasites based on their appearance into either specific blood-stage or abnormal and/or dead. Images were taken with a Leica IC50 HD camera attached to a Leica DM750 microscope. Nanoluciferase (Nluc) bioluminescence was measured from the Hyp1-Nluc parasites by lysing the parasite culture 1:5 with 1x Nano-Glo Lysis Buffer (Promega, USA), and the addition of 1:1000 Nano-Glo (Promega). The bioluminescent signal was measured using a CLARIOstar luminometer (BMG Labtech) at 3000 gain for 1 s/well.

Synchronous Hyp1-NLuc parasites at 28 hours post invasion (hpi) were washed in PBS and diluted to a final density of 1% haematocrit in a 96 well microplate with NPP inhibitors at various concentrations along with a DMSO vehicle control for 20 min at room temp. 10 μL of drug-treated parasites were then transferred to a Greiner Lumitrac microplate and placed in a CLARIOstar luminometer (BMG labtech). To each well, 40 μL of sorbitol buffer (280 mM sorbitol, 20 mM Na-HEPES, 0.1 mg/ml BSA, pH 7.4), containing Nano-Glo substrate (Promega, 1:1000 dilution) was injected and luminescence recorded at 3 minute intervals with the gain set to 2500. Where indicated, 40 μL PBS containing 1:1000 Nano-Glo substrate was used in place of sorbitol as a no-lysis control. The drug concentrations expressed are those after sorbitol dilution. For initial validation, RLU at 15 and 28 minutes was expressed as a percentage relative to the average RLU of DMSO controls. The mean and standard deviation of 48 total wells (16 wells per plate on three separate days) for each drug concentration were used to calculate Z′ values according to the equation17 (link):

The assay was adapted from a previously described method [69 (link)] and utilised ex-Nluc parasites that had been tightly synchronised using 25 nM ML10. Ring-staged parasites (0 to 4 hours postinvasion) were diluted in 96-well U-bottom plates to 1%, 0.1%, and 0.01% parasitemia for cycle one, cycle two, and cycle three, respectively, each with 3 technical replicates. The parasites were then distributed into serially diluted MMV291, azithromycin, and chloroquine from 10 μM, 0.5 μM, and 0.2 μM, respectively, in a 2-step dilution. Parasites were incubated with the compounds for approximately 40 hours until they reached the late-trophozoite to early-schizogony stage and cycle 1 plates were frozen. The remaining plates were washed 3 times before pellets were resuspended to a final volume of 100 μL. The plates were then incubated at 37°C for a further 48 hours before cycle 2 plates were frozen. Cycle 3 plates were grown for a further 48 hours before also being frozen. At the completion of the assay, plates were thawed and the Nluc signal was quantified by adding 5 μL of whole iRBCs to 45 μL of 1 × Nanoglo Lysis buffer with 1:1,000 NanoGlo substrate (Promega) in a white luminometer 96-well plate. A CLARIOstar luminometer (BMG Labtech) was used to measure relative light units (RLUs) and growth was normalised to 0.1% DMSO.

P. falciparum RhopH2-HAglmS and 3D7 infected erythrocytes expressing Hyp1-Nluc were treated with 0–1 mM GlcN when at the trophozoite stage (28–36 hours post invasion) and were then grown for a further 48 h until the parasites were trophozoites again. After washing the parasitized erythrocytes twice in PBS, 10 μL of cell suspension (at 1% hematocrit and 1% parasitemia) were dispensed in triplicate into a white 96 well microplate and loaded into a Clariostar luminometer (BMG Labtech). To each well, 40 μL of sorbitol lysis buffer containing the NanoGlo (Promega) substrate (280 mM sorbitol, 20 mM Na-HEPES, 0.1 mg/ml BSA, pH 7.4, Nano-Glo (1:1000 dilution) was added and the relative light units (RLU) measured every 3 min for up to 1 h with gain set to 2500. The RLU increased over time as more cells lysed and released nanoluciferase. The rate of increase in RLU per min (RLU/min) was determined with GraphPad Prism software^{52 (link)}. A value of 100% lysis is defined as the RLU/min of infected erythrocytes not treated with GlcN (full NPP function) in sorbitol lysis buffer. A value of 0% lysis is defined as the RLU/min of 10 µL parasites in 40 µL of non-lytic PBS containing NanoGlo substrate (1:1000).

Based on the results of viability and FC, the applied concentration of 75 and 500 µg/mL BV at 0 and 24 h were considered for testing metabolic activity. BacTiter-Glo™ Microbial Cell Viability Assay (Promega, USA) and a CLARIOstar Luminometer (BMG Labtech, Germany) were used for the quantitation of the ATP present in bacterial cell culture. The changes in metabolic activity of treated cells were assessed based on the reduction of relative light unit (RLU) in relation to control cells. The BacTiter-Glo™ Microbial Cell Viability Assay was prepared according to manufacturer guidelines. A 100µL aliquot from each treated-cell culture was mixed with an equal volume of BacTiter-Glo™ reagent in triplicate and incubated for 5 min at 150 rpm shaking. After incubation, the luminescence of samples was immediately measured with a Luminometer and analysed using MARS data analysis software.