Abstract
Several cellular mechanisms affect nuclear morphology which can therefore be used to assess certain processes. Here, we present an analytic tool to quantify the number of cells in a population that present characteristics of senescence, apoptosis or nuclear irregularities through nuclear morphometric analysis. The tool presented here is based on nuclear image analysis and evaluation of size and regularity of adhered cells in culture. From 46 measurements of nuclear morphometry, principal component analysis filtered four measurements that best separated regular from irregular nuclei. These measurements, namely aspect, area box, radius ratio and roundness were combined into a single nuclear irregularity index (NII). Normal nuclei are used to set the parameters for a given cell type, and different nuclear phenotypes are separated in an area versus NII plot. The tool was validated with β-gal staining for senescence and annexin or caspases inhibitor for apoptosis as well as several treatments that induce different cellular phenotypes. This method provides a direct and objective way of screening normal, senescent, apoptotic and nuclear irregularities which may occur during failed mitosis or mitotic catastrophe, which may be very useful in basic and clinical research.
Highlights
The nucleus corresponds to approximately 10% of the cellular volume and, due to its nuclear envelope, presents a round shape and a well-defined and regular surface under normal conditions in vitro
Images were analyzed with the Image Pro Plus 6.0 (IPP6) software and 46 parameters of nuclear size, shape and marking were produced for a large population of cells
These parameters were analyzed with principal component analysis (PCA) and the features Aspect (Asp), Area box (Arbx), Radius ratio (Rr) and Roundness (Rou) were combined in an index called Nuclear Irregularity Index (NII = Asp2Arbx+Rr+Rou)
Summary
The nucleus corresponds to approximately 10% of the cellular volume and, due to its nuclear envelope, presents a round shape and a well-defined and regular surface under normal conditions in vitro. A tool was proposed for the detection of MC based on videomicroscopy of cells expressing markers of chromatin and centrosomes [3] This tool requires specific equipment and is rather difficult to set up and analyze. Induction of MC by small molecules or specific inhibition of DNA damage-activated signaling may increase cytotoxicity in cancer cells [8,9] In these cases, co-occurrence of senescence, MC and apoptosis is clinically desirable when compared to the occurrence of only one of these processes [10]. Irregularities in a cell population in vitro based on nuclear morphology
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