| Author | Delvoss, Cleyson Mathias Morais | |
| Author | Inoue, Alexandre Haruo | |
| Author | Silva, Rosiane Valeriano | |
| Author | Fragoso, Stênio Perdigão | |
| Author | Eger, Iriane | |
| Access date | 2024-12-07T18:44:59Z | |
| Available date | 2024-12-07T18:44:59Z | |
| Document date | 2024 | |
| Citation | DELVOSS, C. M. M. et al. Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites. Memórias do Instituto Oswaldo Cruz, v. 119, n. e230223 p. 1-8, 2024. | en_US |
| ISSN | 1678-8060 | en_US |
| URI | https://www.arca.fiocruz.br/handle/icict/67461 | |
| Language | eng | en_US |
| Publisher | Instituto Oswaldo Cruz | en_US |
| Rights | open access | en_US |
| Subject in Portuguese | Atividade tripanocida | en_US |
| Subject in Portuguese | Trypanosoma cruzi | en_US |
| Subject in Portuguese | GFP | en_US |
| Subject in Portuguese | Triagem de drogas | en_US |
| Title | Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites | en_US |
| Type | Article | en_US |
| DOI | 10.1590/0074-02760230223 | |
| 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. | en_US |
| 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. | en_US |
| Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Pesquisa em Apicomplexa. Curitiba, PR, Brasil. | en_US |
| Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Pesquisa em Apicomplexa. Curitiba, PR, Brasil. | en_US |
| Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Molecular e Sistêmica de Tripanossomatídeos. Curitiba, PR, Brasil. | en_US |
| Affilliation | Universidade Estadual de Ponta Grossa. Laboratório de Biologia Celular e Protozoologia. Ponta Grossa, PR, Brasil. | en_US |
| Subject | Trypanocidal activity | en_US |
| Subject | Trypanosoma cruzi | en_US |
| Subject | GFP | en_US |
| Subject | Drug screening | en_US |
| DeCS | Trypanosoma cruzi | en_US |
| DeCS | Técnicas Citológicas | en_US |
| DeCS | Proteínas de Fluorescência Verde | en_US |
