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      Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue Assay

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          Abstract

          Background

          Whatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures. However, the literature still lacks studies regarding the reliability of the TB assay in terms of assessment of its repeatability and reproducibility.

          Methods

          We compared the TB assay's measurements obtained by two biologists who analysed 105 different samples in double-blind for a total of 210 counts performed. We measured: ( a) the repeatability of the count performed by the same operator; ( b) the reproducibility of counts performed by the two operators.

          Results

          There were no significant differences in the results obtained with 2D and 3D cell cultures: we estimated an approximate variability of 5% when the TB assay was used to assess the viability of the culture, and a variability of around 20% when it was used to determine the cell population density.

          Conclusions

          The main aim of this study was to make researchers aware of potential measurement errors when TB is used with a haemocytometer for counting and viability measurements in 2D and 3D cultures. We believe that these results can help researchers to determine whether the expected reliability of the TB assay is compliant with their applications.

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          Most cited references34

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          Spheroid Culture of Mesenchymal Stem Cells

          Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.
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            EVALUATION OF THE TRYPAN BLUE TECHNIQUE FOR DETERMINATION OF CELL VIABILITY.

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              An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide.

              A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.
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                Author and article information

                Contributors
                +39 0543739921 , filippo.piccinini@irst.emr.it
                anna.tesei@irst.emr.it
                chiara.arienti@irst.emr.it
                alessandro.bevilacqua@unibo.it
                Journal
                Biol Proced Online
                Biol Proced Online
                Biological Procedures Online
                BioMed Central (London )
                1480-9222
                20 July 2017
                20 July 2017
                2017
                : 19
                : 8
                Affiliations
                [1 ]ISNI 0000 0004 1755 9177, GRID grid.419563.c, , Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, ; Via Piero Maroncelli 40, 47014 Meldola, FC Italy
                [2 ]ISNI 0000 0004 1757 1758, GRID grid.6292.f, Advanced Research Center on Electronic Systems “Ercole De Castro” (ARCES), , University of Bologna, ; Via Toffano 2/2, 40125 Bologna, Italy
                [3 ]ISNI 0000 0004 1757 1758, GRID grid.6292.f, Department of Computer Science and Engineering (DISI), , University of Bologna, ; Viale Risorgimento, 2, 40136 Bologna, Italy
                Author information
                http://orcid.org/0000-0002-0371-7782
                Article
                56
                10.1186/s12575-017-0056-3
                5518102
                28814944
                12caa73e-6e22-4f86-b583-8baa2e2838bf
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 4 March 2017
                : 2 June 2017
                Categories
                Methodology
                Custom metadata
                © The Author(s) 2017

                Life sciences
                microscopy,oncology,cell viability,haemocytometer,statistical analysis
                Life sciences
                microscopy, oncology, cell viability, haemocytometer, statistical analysis

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