Cytochalasin D vs Nocodazole: Livecyte exposes dramatic differences on a cellular and population level, all label free

Mitosis is a crucial biological process that takes place in all eukaryotic cells and involves the equal segregation and division of a parent cell into two genetically identical daughter cells. Changes to the cell cycle where there is irreversible arrest of cell proliferation have been shown to lead to aging and age-related disease, whereas uncontrollable cell division is linked to cancer.

What is the problem we are solving?

The detection of mitosis events and quantification of their properties is of particular interest in a number of areas, especially oncology where drugs are designed to inhibit cell division and cell cycle progression. To fully understand the protective effects of these compounds, an accurate analytical method is needed to study their actions at different stages of the cell cycle.

Studying the process of mitosis is currently very challenging. Current methods that exist to measure mitosis usually rely on fluorescent labelling of cell mitotic spindles or the manual analysis of brightfield or phase contrast images which use relatively high light levels.

Why is that a problem?

Whilst fluorescent labels can enhance contrast and potentially open the door to automated segmentation and analysis routines, the process and relatively high light levels required can ultimately perturb the natural function and division of cells. The manual analysis of brightfield or phase contrast images is also very time-consuming and subject to human bias.

How does Livecyte solve this?

Livecyte’s primary imaging modality is a quantitative phase imaging technique called ptychography. It produces high-contrast label-free images without the need for fluorescent labels. Coupled with time-lapse imaging and cell tracking algorithms, Livecyte provides users with the tools to monitor mitotic differences over time, independently and in response to external stimuli. Users can see not only how their treatment is affecting the number of mitosis events but also event duration. This provides key insights into the anti-proliferative nature of a treatment, as well as its mode of action on the cell cycle.

A recent study sought to measure and contrast specific cell cycle changes in mitosis between different concentrations of two anti-cancer drugs (cytochalasin D and nocodazole), as compared to untreated cells, through Livecyte’s label-free imaging and analysis tools. This experiment exposed that proliferation alone is far from a reliable indicator of cell cycle behaviour, along with insight into a dramatic dose dependence of these drugs where underdosing is likely to be catastrophic. By quantifying the effect of two cancer treatments on the number of mitosis events and mitotic time, Livecyte enabled insights into how these treatments impacted cell cycle progression. Multi-parametric analysis at a population level view via the Proliferation and Mitosis dashboards, as well as single-cell investigation, allowed a full comparison of cell behaviour in response to the two cell cycle inhibitors and gave a clear quantifiable differentiation between their mechanisms of action.

Figure 1: a) Plot of Confluence and b) Cell Count over time for cells treated with cytochalasin D (300 ng/mL in orange, 9 ng/mL in pink) and untreated control (Bright Pink) and c) histogram plot illustrating median Cell Doubling Times. d) Plot of Confluence and e) Cell Count over time for cells treated with nocodazole (100 µM in blue, 1.23 µM in pink) and untreated control (Bright Pink) and f) histogram plot illustrating median Cell Doubling Times for these cells.

Livecyte’s quantitative phase imaging mode and Analyse software can be utilised to automatically extract a wealth of single cell metrics in the form of proliferation and mitosis data, label-free. In combination with an intuitive workflow, Livecyte enables users to reliably examine the pathways that regulate cell cycle and division in order to enhance the understanding of disease states, from a single assay.

For more information, contact us: +61 2 9541 3500 or enquiries@atascientific.com.au.

Reference

1. Phasefocus.com. 2022. Label-free Analysis of Mitosis Assays [online]. Available at: https://www.phasefocus.com/resources/app-notes/label-free-analysis-mitosis-assays [Accessed 25 May 2022].