title: Trypan blue exclusion assay by flow cytometry.
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.
authors.affiliation: Laboratório de Imunologia, Departamento de Farmácia, Universidade Federal dos Vales do Jequitinhonha e Mucuri and Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil.
authors.forename: B A
authors.initials: BA
authors.lastname: Avelar-Freitas
authors.affiliation: Laboratório de Imunologia, Departamento de Farmácia, Universidade Federal dos Vales do Jequitinhonha e Mucuri and Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil.
authors.forename: V G
authors.initials: VG
authors.lastname: Almeida
authors.affiliation: Departamento de Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
authors.forename: M C X
authors.initials: MC
authors.lastname: Pinto
authors.affiliation: Departamento de Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
authors.forename: F A G
authors.initials: FA
authors.lastname: Mourão
authors.affiliation: Departamento de Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
authors.forename: A R
authors.initials: AR
authors.lastname: Massensini
authors.affiliation: Centro de Pesquisas René Rachou, Fundação Osvaldo Cruz, Belo Horizonte, MG, Brasil.
authors.forename: O A
authors.initials: OA
authors.lastname: Martins-Filho
authors.affiliation: Laboratório de Imunologia, Departamento de Farmácia, Universidade Federal dos Vales do Jequitinhonha e Mucuri and Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil.
authors.forename: E
authors.initials: E
authors.lastname: Rocha-Vieira
authors.affiliation: Laboratório de Imunologia, Departamento de Farmácia, Universidade Federal dos Vales do Jequitinhonha e Mucuri and Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Diamantina, MG, Brasil.
authors.forename: G E A
authors.initials: GE
authors.lastname: Brito-Melo
mesh_terms: D017252:CD3 Complex / Q000097:blood*; D002452:Cell Count; D002462:Cell Membrane / Q000502:physiology; D002469:Cell Separation; D002470:Cell Survival; D005434:Flow Cytometry / Q000592:standards*; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D006801:Humans; D016130:Immunophenotyping; D007202:Indicators and Reagents / Q000592:standards; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000592:standards; D011361:Professional Competence; D011419:Propidium / Q000592:standards; D012710:Serum Albumin, Bovine / Q000592:standards; D013194:Staining and Labeling; D014343:Trypan Blue; D055815:Young Adult
chemical_list: D017252:CD3 Complex; D007202:Indicators and Reagents; D046912:Multiprotein Complexes; D012710:Serum Albumin, Bovine; D011419:Propidium; D016650:Fluorescein-5-isothiocyanate; D014343:Trypan Blue