![]() In recent years, software programs that allow for the automated analyses of images have also been used to evaluate lipid content. Although ImageJ allows the development of macros to automate a series of ImageJ commands for complex analyses, standard components of the software do not allow for proper identification of individual lipid droplets. The ImageJ free software is one of the most commonly used image analysis platforms for lipid content evaluation. Current analytical methods use basic software that allows for deconvolution of images based on color detection to quantify total lipid content. 2018) or digital quantification (for Oil Red O or BODIPY) ( Park et al. Methods to quantify intracellular lipid accumulation after differentiation range from visual observation of stain (most often Oil Red O or Nile Red) on a culture plate ( Prieto-Echague et al. ![]() After lipid staining, visualization of lipid droplets is most routinely carried out by transmission light microscopy or fluorescence microscopy, with fluorescence probes being more sensitive to detection ( Klymchenko 2017). BODIPY493/503 is more selective for lipid droplet detection, as the poor solubility of Oil Red O can damage or result in the fusion of lipid droplets during the ethanol/isopropanol staining step ( Fukumoto and Fujimoto 2002). ![]() 2018), two of the most common probes are Oil Red O, a lysochrome-diazo dye that stains lipids with a red color at 518 nm, and BODIPY493/503, an organoboron fluorescent dye. While there are over 50 different probes to detect lipid droplets ( Fam et al. 2019).Ĭurrent techniques to detect intracellular lipid accumulation after differentiation include the use of dyes that will stain for neutral triglycerides and lipids. During the differentiation process, cells begin to accumulate lipid droplets leading to the formation of mature adipocytes with a specific transcriptomic profile, including increased PPARγ, FABP4, and adiponectin expression ( Lee JE et al. Preadipocytes are induced to differentiate in vitro for 8 to 12 days during which cells are exposed to a differentiation cocktail that may contain insulin, peroxisome proliferator-activated receptor gamma (PPARγ) agonists, and/or 3-isobutyl-1-methylxanthine (IMBX), among others ( Zhao et al. Studies to understand adipogenesis often use the preadipocyte mouse cell line 3T3-L1. Understanding how these factors can alter adipogenesis can help provide insights into strategies to prevent adipose tissue accumulation resulting in metabolic disorders and/or obesity ( Ghaben and Scherer 2019). This is a tightly controlled process that may be altered by several factors, including nutrition, stress, as well as environmental factors ( Ghaben and Scherer 2019). In conclusion, this novel image analysis tool can provide with a more precise evaluation of lipid droplet and adipogenesis dysregulation, a critical need in the understanding of metabolic disorders.Īdipogenesis is the process by which mesenchymal stromal cells or committed preadipocytes differentiate into mature adipocytes. CellProfiler streamlines the lipid droplet phenotypic analysis of adipocytes compared to more traditional analysis methods. A clustering analysis is also possible using CellProfiler which allows for the quantification of total lipid content per individual adipocyte to provide insight into single-cell responsiveness to adipogenic stimuli. Our results show that CellProfiler is able to accurately identify a greater number of lipid droplets compared to ImageJ. For ImageJ, we used an already developed macro designed to identify particles and quantify their area, and for CellProfiler, we developed a new analysis pipeline. For the lipid droplet analysis, we used two approaches, the free online computer software of reference, ImageJ, and another free online computer software, CellProfiler. Therefore, the aims of this study were to develop an accurate, standardized approach to quantify lipid droplet size of mature adipocytes and a clustering approach to analyze the total lipid content per adipocyte. Nutrition, stress, or chemical exposure can dysregulate adipogenic differentiation and lipid metabolism. However, imaging tools for evaluating intracellular lipid droplets remain at their infancy. During differentiation, neutral lipids that accumulate in adipocytes can be detected using stains and used as an index of cell differentiation. Adipogenic differentiation is the process by which preadipocytes become mature adipocytes, cells that store energy and regulate metabolic homeostasis. ![]()
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