In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites
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The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.
KeywordsResazurin Viability Percentage Contact Lens Wearer Acanthamoeba Keratitis Acanthamoeba Castellanii
We wish to thank Mrs. Isabel Trabado and Mrs. Cristina Miguel (Cell Culture UnitCAI Medicina y Biología de la Universidad de Alcalá) for the technical assistance, Dr. Javier Martínez for statistical assistance and to Dr. Ann-Karolin Scheu for language assistance.
This work received financial support from MICINN (CTQ2011-23245), CAM (S2010/BMD-2351) and CIBER-BBN. Ms. Heredero-Bermejo wishes to thank the Ministerio de Educación y Ciencia for the fellowship FPU ref. AP2010-1471.
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