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Automated quantification of vomeronasal glomeruli number, size, and color composition after immunofluorescent staining.

ABSTRACT: Glomeruli are neuropil-rich regions of the main or accessory olfactory bulbs (AOB) where the axons of olfactory or vomeronasal neurons and dendrites of mitral/tufted cells form synaptic connections. In the main olfactory system, olfactory sensory neurons (OSNs) expressing the same receptor innervate 1 or 2 glomeruli. However, in the accessory olfactory system, vomeronasal sensory neurons (VSNs) expressing the same receptor can innervate up to 30 different glomeruli in the AOB. Genetic mutation disrupting genes with a role in defining the identity/diversity of olfactory and vomeronasal neurons can alter the number and size of glomeruli. Interestingly, 2 cell surface molecules, Kirrel2 and Kirrel3, have been indicated as playing a critical role in the organization of axons into glomeruli in the AOB. Being able to quantify differences in glomeruli features, such as number, size, or immunoreactivity for specific markers, is an important experimental approach to validate the role of specific genes in controlling neuronal connectivity and circuit formation in either control or mutant animals. Since the manual recognition and quantification of glomeruli on digital images is a challenging and time-consuming task, we generated a program in Python able to identify glomeruli in digital images and quantify their properties, such as size, number, and pixel intensity. Validation of our program indicates that our script is a fast and suitable tool for high-throughput quantification of glomerular features of mouse lines with different genetic makeup.

SUBMITTER: Bahreini Jangjoo S

PROVIDER: S-EPMC8502234 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMultipotent mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues and are considered as attractive candidates for the development of cell-based regenerative therapies. Currently, there are more than 200 clinical trials involving the use of MSCs for a wide variety of indications. However, variations in their isolation, expansion, and particularly characterization have made the interpretation of study outcomes or the rigorous assessment of therapeutic efficacy difficult. An unbiased characterization of MSCs is of major importance and essential to guaranty that only the most suitable cells will be used. The development of standardized and reproducible assays to predict MSC potency is therefore mandatory. The currently used quantification methodologies for the determination of the trilineage potential of MSCs are usually based on absorbance measurements which are imprecise and prone to errors. We therefore aimed at developing a methodology first offering a standardized way to objectively quantify the trilineage potential of MSC preparations and second allowing to discriminate functional differences between clonally expanded cell populations.MethodMSCs originating from several patients were differentiated into osteoblasts, adipocytes, and chondroblasts for 14, 17, and 21 days. Differentiated cells were then stained with the classical dyes: Alizarin Red S for osteoblasts, Oil Red O for adipocytes, and Alcian Blue 8GX for chondroblasts. Quantification of differentiation was then performed with our newly developed digital image analysis (DIA) tool followed by the classical absorbance measurement. The results from the two techniques were then compared.ResultQuantification based on DIA allowed highly standardized and objective dye quantification with superior sensitivity compared to absorbance measurements. Furthermore, small differences between MSC lines in the differentiation potential were highlighted using DIA whereas no difference was detected using absorbance quantification.ConclusionOur approach represents a novel method that simplifies the laboratory procedures not only for the quantification of histological dyes and the degree of differentiation of MSCs, but also due to its color independence, it can be easily adapted for the quantification of a wide range of staining procedures in histology. The method is easily applicable since it is based on open source software and standard light microscopy.

Dataset Information

Automated quantification of vomeronasal glomeruli number, size, and color composition after immunofluorescent staining.

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