Combination of Free Software, Light Microscopy and Non-specific Dyes for a High-throughput Analysis in Live/Dead Cell Count

Background: Cellular viability studies are important in many different fields of cell biology research and are based on estimates of live and dead cells from a specific cellular population. Recent progress in technology has brought important advances in the analysis of cellular viability, mainly reducing the time of analysis and sample preparation; however, the costs for equipment and reagents still limit the feasibility of conducting those studies. The purpose of the present study was to use free software, non-specific dyes, and light microscopy to establish a reproducible image analysis test and to compare the results with an established method. Method: 3T3 fibroblast cell line was exposed to different concentrations of dimethylsulfoxide (DMSO) for 24 h, and cellular viability was estimated by the proposed technique (image analysis performed with free software) and with the methylthiazol-tetrazolium salt (MTT) assay. A Bland-Altman analysis was applied to compare the methodologies. Results: Image analysis by CellProfiler®/CellProfiler® Analyst was compared to MTT by Bland-Altman test, and the two methodologies were considered equivalent. Cellular viability decreased in a dose-dependent manner with increasing doses of DMSO, and both methodologies were capable of distinguishing between live and dead cells, producing comparable results (with bias = 1.3550) evaluated by the Bland-Altman analysis. Conclusion: The proposed image analysis can be used as a simple, rapid, and low-cost technology for high throughput analysis of live-dead cell differentiation.