Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images

Abstract

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k = 3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

Integration of high-content fluorescence imaging into the metabolic flux assay reveals insights into mitochondrial properties and functions

“…2G ), demonstrating another utility for our imaging platform. Finally, we observed increased OCR in T3M4 cells that have been treated with taxol, a previously described feature of taxol administration 20 ( Fig. 2H ).…”

“…Nuclei staining intensity can be used to infer the stage of the cell cycle 20,21 . Therefore, we sought to determine if we could integrate this analysis to the XF-Cytation5 platform.…”

“…Recent adaptations to the metabolic flux assay have incorporated nuclei fluorescent staining (Hoechst dye) and subsequent imaging with the BioTek Cytation 5 (BioTek, VT, USA) to more adequately control for cell number 19 . Herein, we optimize and extend this concept, as nuclei staining with DAPI or Hoechst dyes can also be applied to determine cell-cycle analysis 20,21 ; an important cellular characteristic when examining drug responses in high-throughput screens. Importantly, mitochondrial bioenergetics have been previously shown to coordinate with cell cycle dynamics 22,23 , further supporting the use of nuclei counterstaining in conjunction with the metabolic flux assay.…”

Triptolide Induces Apoptosis Through Fas Death and Mitochondrial Pathways in HepaRG Cell Line

“…The use of the fluorescent dye DAPI stain for DNA can aid in visual analysis of nuclear morphological changes ( Ferro et al, 2017 ). HepaRG cells (4.0 × 10 5 cells/well) were seeded and treated with different concentrations of triptolide (0, 100, 200, and 400 nM) for 24 h. The cells were collected and washed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at room temperature.…”

The application of impedance measurement to assess biofilm development on technical materials used for water supply system construction

“…For this purpose, 1 µl of dye was applied onto the test material and then it was incubated in the dark for 20 minutes, then the samples were viewed under a microscope. Live bacterial cells dyed with DAPI are fluorescent blue [13].…”