Microbeta2 counter

Cells were seeded in 96‐well microtiter plates (5 × 10³ C2 or NI‐1 cells/well; 15 × 10⁴ primary MCs/well) and incubated in control medium, vehicle control or various concentrations of HR1 antagonists (0.1–75 µM) at 37°C for 48 hr. Since the concentrations of HR1 antagonists required to block MCT cell growth were rather high, we were interested to identify drug‐combinations in which the concentrations of the individual drugs could be reduced to a pharmacologically meaningful range. Therefore, we tested combinations of HR1 antagonists and TKIs at suboptimal concentrations (20%–30% maximal proliferation reduction) to evaluate cooperative effects. After incubation, 0.5 µCi of ³H‐thymidine was added (37°C, 16 hr). Thereafter, cells were harvested on filter membranes (Perkin Elmer, Waltham, MA, USA) in a Filtermate 196 harvester (Packard Bioscience, Meriden, CT, USA) and the bound radioactivity was measured in a MicroBeta² Counter (Perkin Elmer). All experiments were performed in triplicates and experiments with cell lines were repeated at least three times.

The viability of the parasites following chemical attenuation was assessed using the [³H]-hypoxanthine growth inhibition assay. Chemically attenuated ring-stage parasites (2% haematocrit) were added to 96-well flat-bottomed plates (100 μl per well) in quadruplicate. Unattenuated ring-stage parasites and unparasitised red blood cells (uRBC) at 2% haematocrit were used as positive and background controls respectively. Plates were placed in a 37 °C incubator with 5% O₂, 5% CO₂, and 90% N₂. The assay duration was 48 h with [³H]-hypoxanthine (0.2 μCi/well) added from the start of the experiment. Following incubation, plates were frozen, then subsequently thawed and harvested onto glass fibre mats (Perkin Elmer, Australia) using a Filtermate cell harvester (Perkin Elmer). Radioactivity was measured using a Microbeta² counter (Perkin Elmer). The remainder of the packed cells from the vaccine were placed in culture, and after 1 week, 2 weeks and 3 weeks of culture, cells were harvested and evaluated according to incorporation of [³H]-hypoxanthine. Twice a week, fresh uRBC were added into the cultures and the medium changed. No growth was observed, as measured by lack of [³H]-hypoxanthine incorporation, compared to unattenuated Pf 7G8 control samples that were cultured in parallel.

SK-MEL-28 cells (1 × 10⁵/well) were incubated with [³H]-L-glutamine (400 nM; PerkinElmer) in RPMI media (Life Technologies) without L-glutamine for 15 min at 37°C in the presence or absence (Control; vehicle) of each inhibitor as previously published (van Geldermalsen et al., 2016 (link)). Cells were collected and transferred to filter paper using a 96-well plate harvester (Wallac PerkinElmer), dried, exposed to scintillation fluid and counts measured using a liquid scintillation counter as previously published (MicroBeta² Counter, PerkinElmer) (Wang et al., 2014 (link)).

In the following
experiments, the MCF-7, HEK-hLAT1, and HEK-MOCK cells were cultured,
seeded, and preincubated as described above. The HBSS was removed,
and the ability of the compounds to inhibit the uptake of a known
LAT1 substrate, [¹⁴C]-L-leucine (PerkinElmer, Waltham,
MA, USA), was studied by incubating the cells at RT for 10 min in
uptake buffer pH 7.4 (250 μL) containing 0.76 μM (0.1
mCi/mL) of [¹⁴C]-L-leucine and 0.025–1800 μM
of the studied compound (or HBSS as blank). After incubation, the
reaction was stopped with ice-cold HBSS, and the cells were washed
two times with ice-cold HBSS. The cells were then lysed with 250 μL
of 0.1 M sodium hydroxide, the lysate was mixed with 1.0 mL of Emulsifier
safe cocktail (PerkinElmer, Waltham, MA, USA), and the radioactivity
was measured with a liquid scintillation counter (MicroBeta² counter, PerkinElmer, Waltham, MA, USA). The inhibition of [¹⁴C]-L-leucine in the presence of the studied compounds as
compared to the control (HBSS) was calculated as percentages (%).