Short technical reportA malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue
Graphical abstract
. An efficient and reliable gametocytocidal drug screening assay was developed using cell viability marker, alamarBlue.
Research highlights
► Inhibition data from alamarBlue assays are similar to traditional optical assays. ►Assay time for 96 samples is decreased from 10 h to 1 min. ► Reproducibility is increased. ► Any parasite line, including field isolates, can be tested for drug sensitivity.
Section snippets
Assay optimization
AlamarBlue was first screened to ensure that it did not interfere with gametocyte development or the gametocytocidal activity of proteasome inhibitor, epoxomicin [14]. Gametocyte (3D7 strain) cultures were set up in complete media (RPMI 1640 supplemented with l-Glutamine, 25 mM HEPES, 50 μg ml−1 hypoxanthine (KD Biomedical), 10 μg ml−1 gentamicin, 0.25% NaHCO3, and 10% human serum) as described [15] and the parasites were treated with N-acetyl glucosamine (NAG) from day 9 to day 11 to eliminate
Final assay conditions
Once the basic conditions were established for the alamarBlue assay it was directly compared with the current standard assay, optical microscopy of Giemsa-stained smears. The epoxomicin dose response was used for comparison. For the optical assay, 10 μl of DMSO alone or in combination with the indicated concentration of epoxomicin were added to NAG-treated gametocyte cultures (1.45% gametocytemia (stages III–V)/3% hematocrit on day 13). Giemsa-stained smears were prepared at 72 and 96 h after
Gametocytocidal activity of common anti-malarial drugs
As a further test, the alamarBlue assay was used to screen the gametocytocidal activity of a panel of current anti-malarial drugs, pyrimethamine, chroloquine, quinine, mefloquine, dihydroartemisinine (DHA) and primaquine (Fig. 2). Consistent with previous reports pyrimethamine, chloroquine, quinine or mefloquine did not show gametocytocidal activity, while primaquine showed a reduction of 69.4 ± 17.2% activity at 10 μM. DHA, the active metabolite of artemisinin was the most effective against late
Acknowledgements
This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Dr. S. Desai for use of the fluorescent plate reader and Dr. B. Morahan for critical reading of the manuscript.
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2019, Current Opinion in Chemical BiologyCitation Excerpt :Towards an improved signal-to-noise ratio, required for HTS, several groups have developed colorimetric readouts for gametocyte viability. This includes parasite lactate dehydrogenase (pLDH) [52] and alamarBlue [53] as indicators of metabolic activity. Two recent large-scale screens are also worth highlighting, including the use of acridine orange (AO) to measure gametocytaemia and rounding-up post-activation as a marker of viability, adapted to 384-well format from researchers at the Istituto Superiore di Sanità in Rome [54] and, most recently, the Saponin-lysis Sexual Stage Assay (SaLSSA) from the University of California San Diego School of Medicine. [11••].
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