| Author | Freitas, Bethânia Alves de Avelar | |
| Author | Almeida, Valéria Gomes de | |
| Author | Pinto, Mauro Cunha Xavier | |
| Author | Mourão, Flávio Afonso Gonçalves | |
| Author | Massensini, Andre Ricardo | |
| Author | Martins Filho, Olindo Assis | |
| Author | Vieira, Etel Rocha | |
| Author | Melo, Gustavo Eustáquio Brito Alvim de | |
| Access date | 2018-10-19T19:16:20Z | |
| Available date | 2018-10-19T19:16:20Z | |
| Document date | 2014 | |
| Citation | FREITAS, Bethânia Alves de Avelar et al. Trypan blue exclusion assay by flow cytometry. Braz J Med Biol Res. , v. 47, n. 4, p. 307-15, 2014. | pt_BR |
| ISSN | 0100-879X | pt_BR |
| URI | https://www.arca.fiocruz.br/handle/icict/29669 | |
| Language | eng | pt_BR |
| Publisher | Associação Brasileira de Divulgação Científica | pt_BR |
| Rights | open access | pt_BR |
| Subject in Portuguese | Azul tripano | pt_BR |
| Subject in Portuguese | Fluorescência | pt_BR |
| Subject in Portuguese | Citometria de fluxo | pt_BR |
| Subject in Portuguese | Citotoxicidade | pt_BR |
| Title | Trypan blue exclusion assay by flow cytometry | pt_BR |
| Type | Article | pt_BR |
| DOI | 10.1590/1414-431X20143437 | |
| Abstract | Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis. | pt_BR |
| Affilliation | Universidade Federal dos Vales do Jequitinhonha e Mucuri. Departamento de Farmácia. Laboratório de Imunologia. Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil / Universidade Federal dos Vales do Jequitinhonha e Mucuri. Instituto de Ciência e Tecnologia. Diamantina, MG, Brasil. | pt_BR |
| Affilliation | Universidade Federal dos Vales do Jequitinhonha e Mucuri. Departamento de Farmácia. Laboratório de Imunologia. Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil. | pt_BR |
| Affilliation | Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Fisiologia e Farmacologia. Belo Horizonte, MG, Brasil. | pt_BR |
| Affilliation | Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Fisiologia e Farmacologia. Belo Horizonte, MG, Brasil. | pt_BR |
| Affilliation | Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Fisiologia e Farmacologia. Belo Horizonte, MG, Brasil. | pt_BR |
| Affilliation | Fundação Osvaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil. | pt_BR |
| Affilliation | Universidade Federal dos Vales do Jequitinhonha e Mucuri. Departamento de Farmácia. Laboratório de Imunologia. Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil. | pt_BR |
| Affilliation | Universidade Federal dos Vales do Jequitinhonha e Mucuri. Departamento de Farmácia. Laboratório de Imunologia. Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil. | pt_BR |
| Subject | Trypan blue | pt_BR |
| Subject | Fluorescence | pt_BR |
| Subject | Flow cytometry | pt_BR |
| Subject | Cytotoxicity | pt_BR |
| Embargo date | 2020-01-01 | |
