Cell membrane labeling of Eimeria tenella sporozoites with the fluorescent dye PKH-67 GL for tracking parasite-host interactions.
The fluorescent cell linker dye PKH-67 GL was used as a vital stain for sporozoites of Eimeria tenella for tests on viability, invasion of cultured primary chick kidney cells, flow cytometric analysis and fluorescence microscopy. The effect of PKH-67 GL on sporozoites was tested at a range of concentrations of dye and sporozoites. In flow cytometric analysis, 0.5-40x10(-6) M of PKH-67 GL labeled sporozoites to some degree, with the percentage of labeled sporozoites increasing with higher dye concentrations. The optimum concentration was 2x10(-6) M, allowing easy observation by fluorescence microscopy. Morphological changes in the sporozoite at concentrations greater than 5x10(-6) M were accompanied by loss of viability according to a propidium iodide inclusion assay. Sporozoite penetration of primary chick kidney cells was unaffected by the optimal level of 2x10(-6) M, allowing observation of intracellular activities. Overall, the cell linker dye greatly facilitated observation of E. tenella in vitro and in flow cytometric analysis.
Fuller
A L
AL
Department of Poultry Science, University of Georgia, Athens 30602, USA. alfuller@arches.uga.edu
McDougald
L R
LR
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured / Q000469:parasitology; D004305:Dose-Response Relationship, Drug; D016786:Eimeria tenella / Q000254:growth & development* / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006790:Host-Parasite Interactions / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000469:parasitology; D008856:Microscopy, Fluorescence
D005456:Fluorescent Dyes