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https://www.arca.fiocruz.br/handle/icict/67461
IMPROVING IN VITRO SCREENING COMPOUNDS ANTI-TRYPANOSOMA CRUZI BY GFP-EXPRESSING PARASITES
Author
Affilliation
Universidade Estadual de Ponta Grossa. Laboratório de Biologia Celular e Protozoologia. Ponta Grossa, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Pesquisa em Apicomplexa. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Pesquisa em Apicomplexa. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Pesquisa em Apicomplexa. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Molecular e Sistêmica de Tripanossomatídeos. Curitiba, PR, Brasil.
Universidade Estadual de Ponta Grossa. Laboratório de Biologia Celular e Protozoologia. Ponta Grossa, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Pesquisa em Apicomplexa. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Pesquisa em Apicomplexa. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Molecular e Sistêmica de Tripanossomatídeos. Curitiba, PR, Brasil.
Universidade Estadual de Ponta Grossa. Laboratório de Biologia Celular e Protozoologia. Ponta Grossa, PR, Brasil.
Abstract
BACKGROUND Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening.
OBJECTIVES In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability.
METHODS Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry.
FINDINGS The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed.
MAIN CONCLUSIONS Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time consuming microscopic counting of intracellular amastigotes.
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