Trypan blue exclusion assay by flow cytometry

B.A Avelar-Freitas,G.E.A Brito-Melo,V.G Almeida,A.R Massensini,M.C.X Pinto,F.A.G Mourão,E Rocha-Vieira,O.A Martins-Filho

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.

Highlights

Cell viability analysis is a useful tool in various experimental procedures, including those for tumor susceptibility, microbiological resistance, and spontaneous cell death after submission to different experimental conditions [1,2,3,4]
Because the trypan blue (TB)-bovine serum albumin (BSA) complex emits fluorescence, we evaluated whether TB inside cells in the form of complexes with cytoplasmic proteins could present a similar behavior
The number of live and dead cells in an experiment can be estimated by the use of several markers including dyes that intercalate into DNA, reagents that bind to phosphatidylserine (Annexin V), dyes that stain cells that lose membrane selectivity, and amine reactive dyes (UViD, ViViD, GrViD, OrViD: UV, violet, green, and orange fluorescence, respectively)

Summary

Introduction

Cell viability analysis is a useful tool in various experimental procedures, including those for tumor susceptibility, microbiological resistance, and spontaneous cell death after submission to different experimental conditions [1,2,3,4]. The trypan blue (TB) method is a very common assay for evaluating cytotoxicity in experimental investigations [6,7,8] where dead cells absorb TB into the cytoplasm because of loss of membrane selectivity, whereas live cells remain unstained [6]. The relative number of dead and live cells is obtained by optical microscopy by counting the number of stained (dead) and unstained (live) cells using a Neubauer chamber. This conventional TB exclusion assay, when used for a large number of samples, can provide lowprecision results because of the lengthy run time and intensive microscopic examination needed [9]

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