Characterization of Acanthamoeba-microsphere association by multiparameter flow cytometry and confocal microscopy.
BACKGROUND
Acanthamoebae, in common with other protozoa, readily endocytose particulate material, which in turn may lead to the spread of infectious disease.


METHODS
Evaluation and quantification of plain and carboxylate FITC-microsphere association with acanthamoebal trophzoites was undertaken using a combination of flow cytometry and confocal microscopy. Trophozoites from strains and species of Acanthamoeba were exposed to plain and carboxylate FITC-microspheres. Microsphere size and aspects such as trophozoite starvation, maturity, and exposure to metabolic inhibitors were assessed.


RESULTS
All species and strains of Acanthamoeba readily endocytosed plain and carboxylate microspheres. Starving trophozoites significantly increased binding and potential ingestion of microspheres, whereas trophozoites of increasing maturity lost such abilities. Trophozoites showed a significant preference for 2.0- and 3.0-microm-diameter microspheres when compared with other sizes, which in turn could occupy much of the cytoplasm. The physiological inhibitors sodium azide, 2,4-dinitrophenol, and cytochalasin B reduced microsphere association with trophozoites; however, some microspheres still bound and associated with trophozoites after inhibitor exposure, a manifestation of both active and inactive agent involvement in microsphere endocytosis.


CONCLUSIONS
Even though the origins of microsphere binding by acanthamoebal trophozoite remains shrouded, the combination of flow cytometry and confocal microscopy supported synergistic quantification and qualification of trophozoite-microsphere endocytosis.
Life and Health Sciences, Aston University, Birmingham, United Kingdom.
Edward A G
EA
Elloway
Roger A
RA
Bird
Christopher J
CJ
Hewitt
Steven L
SL
Kelly
Stephen N
SN
Smith
D000048:Acanthamoeba / Q000502:physiology*; D000818:Animals; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008018:Life Cycle Stages / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods*; D008863:Microspheres
D005456:Fluorescent Dyes; D016650:Fluorescein-5-isothiocyanate