Journal: Communications Biology

Article Title: A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation

doi: 10.1038/s42003-019-0737-3

Figure Lengend Snippet: a Schematic of the most important building blocks of the construct used for transfection; for details of the complete construct, see methods and the Supplementary Data file . The plasmid contains two fluorescent reporters, GFP driven by the hsp70 promoter and mCherry controlled by the lisp2 promoter. A centromere is included to maintain the construct throughout the life cycle. b Dot plot of the number of small and large exoerythrocytic forms (EEFs) per well in a 96-well plate with s.e.m. from n = 6 wells. Results derive from a side by side comparison of transgenic and wild-type parasites. c Left, P. cynomolgi merosomes (transgenic line) in culture at day 10 post sporozoite invasion. Scale bar, 50 µm. Right, Giemsa staining of red blood cells collected one day post overlay on a liver stage culture containing merosomes shows two ring stage blood forms. d GFP expression (live) in two small forms and one large form at day 6 post inoculation of a primary hepatocyte culture with transgenic P. cynomolgi sporozoites; the same image after fixation and IFA with anti-HSP70 antibodies, and an overlay with DAPI. Scale bars, 25 µm.

Article Snippet: Hepatocytes were seeded into collagen coated 96-well CellCarrier Ultra plates (PerkinElmer) at a density of ~65 × 10 3 cells/well in William’s B medium: William’s E with glutamax containing 10% human serum (A+), 1% MEM nonessential amino acids, 2% penicillin/streptomycin, 1% insulin/transferrin/selenium, 1% sodium pyruvate, 50 μM β-mercapto-ethanol, and 0.05 μM hydrocortisone.

Techniques: Construct, Transfection, Plasmid Preparation, Comparison, Transgenic Assay, Staining, Expressing

Journal: Communications Biology

Article Title: A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation

doi: 10.1038/s42003-019-0737-3

Figure Lengend Snippet: a Images of parasites visualized live by fluorescence microscopy using an Operetta High Content imager. Overviews are shown from stitched images of the same well (96-well plate) at different time points, showing GFP-expressing parasites (upper panel) and mCherry-expressing parasites (lower panel). Parasites from the yellow inset are depicted in b . Scale bar, 500 µm. b Images of a small and a developing form expressing GFP (upper panel) and mCherry (lower panel) at different time points after sporozoite invasion. Yellow arrows mark expression, and grey arrows mark absence of fluorescence. At day 11, individual merozoites can be observed. Scale bar, 50 µm. c Operetta measurements of fluorescent EEF surface areas (left panel), mCherry intensity (middle panel), and GFP intensity (right panel) in individual parasites (blue dots) followed at different days p.i. (post infection) in ten fields of a single well. The bar represents the median values.

Article Snippet: Hepatocytes were seeded into collagen coated 96-well CellCarrier Ultra plates (PerkinElmer) at a density of ~65 × 10 3 cells/well in William’s B medium: William’s E with glutamax containing 10% human serum (A+), 1% MEM nonessential amino acids, 2% penicillin/streptomycin, 1% insulin/transferrin/selenium, 1% sodium pyruvate, 50 μM β-mercapto-ethanol, and 0.05 μM hydrocortisone.

Techniques: Fluorescence, Microscopy, Expressing, Infection