Optimization of assay parameters and characterization of gametocyte-specific GFP reporter line.

<p>(<b>A</b>) Comparison between microscopy and FCM for quantifying parasitemia. Gametocytes were serially diluted and each data point was determined in duplicates by microscopy and analyzed in parallel by FCM. The corresponding <i>R</i>² values are 0.9641 by microscopy and 0.9953 by FCM. (<b>B</b>) The maximum/minimum signal ratios at different gametocyte stages. Stage I–V gametocytes at 0.02% gametocytemia were seeded in 96-well plates and ∼half a million events were counted for each well. The box-and-whisker plot represents the ratio of fluorescence intensity (FI) for control wells to FI of these wells treated with 625 µM of PMQ. (<b>C</b>) Assessment of GFP levels in stage V gametocytes after drug treatment by fluorescence microscopy. Gametocytes emitting strong GFP fluorescence also showed strong staining of mitochondrion with JC-1, indicating of live cells (top panel). Gametocytes showing weaker GFP fluorescence also had weaker JC-1 staining of the mitochondrion, indicating dying or dead gametocytes (middle and bottom panels, respectively).