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    Cell Cytoxicity Assay

Highlights

Use non-invasive time-lapse imaging to quantify cell death without labels, dyes or phytotoxic damage.

  • Perform continuous time-lapse imaging to mitigate the need for time and labour-intensive multiple fixed-point measurements.
  • Reveal the kinetic information of treatments on cell toxicity.
  • Obtain statistically robust measures of cell death by sampling large cell populations.
  • Measure morphological and behavioural profiles of individual cells to investigate modes of cell death.

Introduction

Cytotoxicity is a measure of the ability for compounds/substances to cause cellular death. The cytotoxicity assay is widely used in the pharmaceutical industry and is a fundamental tool in the drug discovery process.

This application note describes the ability of the Phasefocus™ Livecyte™ system to detect cellular death in a non-invasive manner. A distinct advantage in using the Livecyte system is the ability to obtain, over a long period of time, both high contrast and quantitative images, without the requirement of introducing fluorescent labels into the culture. The morphological and quantitative data attainable has the potential to determine the mode of cell death. Furthermore, due to the low illumination power required, the cells remain viable post imaging, allowing for additional downstream analysis.

Cell Culture and Preparation

A549 cells were seeded in glass-bottomed 6-well plates at a density of 105cells/well. The Livecyte system was used to acquire ptychographic images in time-lapse, every 6 minutes over a period of 70 hours. The cells were imaged using a 10x (NA 0.2) objective. The Livecyte incubation environment was maintained at a constant 37 ºC and a constant 5% CO2 in air. Cell death was induced by application of staurosporine across a range of concentrations. Following acquisition, the images were analysed using the Phasefocus Cell Analysis Toolbox™ (CAT) software. Population-based metrics relating to cellular growth/proliferation and cellular death were determined. In addition, individual cell metrics such as morphological and temporal data were investigated.

Identification of Cell Death and Cell Growth

The quantitative output from the Livecyte system (cellular dry mass) enables the determination of the difference between cell death and cell growth. Individual cells were segmented and tracked as they went through either cell growth/mitosis or cell death (Fig. 1). During cell growth the mass of the cell increases until it reaches mitosis, after which it divides into 2 daughter cells that then also undergo cell growth (Fig. 1a). Conversely, during cell death the dry mass does not increase but once the membrane is compromised, the cell begins to lose mass as the membrane ruptures (Fig. 1b).

Calculating the total dry mass of all cells in a field of view can be used to measure both cellular death and cellular growth of the population of cells. Cellular death of A549 cells was induced by application of increasing concentrations of staurosporine (Fig. 2). The effect of staurosporine is clearly evident in Fig. 2a, showing representative images at 0, 36 and 72-hour time-points in both control conditions and following application of 10μM staurosporine. In order to quantify the rate of cell death or proliferation, the relative dry mass (normalised to total dry mass at t0) of the population was calculated at each time point (Fig. 2b). Treating the cells with 10 μM staurosporine resulted in a reduction in relative dry mass (0.65 at 72hours), whereas in the untreated cells an increase is observed as cells grow/proliferate (4.23 at 72hours). Within the same assay the cytostatic effect of 1 μM and the anti-proliferative effect of 100, 10 and 1nM staurosporine is also observed by measuring the dry total dry mass over time (Fig. 2b).

In addition to population based data, the Livecyte system offers the benefit of single cell analysis. By performing this type of analysis, morphological information is available to further investigate the mode of action of staurosporine (Fig. 2c). A549 cells treated with 10 μM staurosporine show an increase in cell sphericity, which is consistent with the morphological changes during cell death. Application 1 μM staurosporine however, showed little increase in total dry mass over the 72hours indicating a cytostatic response together with a reduction in cell sphericity, indicating a change in the cellular phenotype.

Video

Figures

Cell Cytotoxicity

Figure 1. Label-free identification of cellular growth and death. a) Representative images of tracked A549 cell (cell 1) undergoing mitosis into   2 daughter cells (cell 15 and 17) and b) an A549 cell undergoing cell death following treatment with 10 μM staurosporine, together with the quantitative output of dry mass over time.

Cell Cytotoxicity

Figure 2. Effect of staurosporine to a population of cells a) Frames extracted at 0hrs, 36hrs and 72 hrs from a time lapse comparing the effect of a population with staurosporine and a control.          b) Overall population metrics for various concentrations of staurosporine over 72hrs and
 c) Utilising the ability of the Livecyte system to interrogate the parameters of cell culture to a single cell level.

Download the Application Note pdf

Conclusion

This application note demonstrates the effective use of the Phasefocus Livecyte system to image and quantify cellular toxicity in a non-invasive manner. This holds key advantages, as it does not require the use of fluorescent labels nor does it induce any phototoxic effects on the cells under measurement. Consequently, the behaviour of the cell culture during the time course is unrelated to any unintended influence from the imaging process or from the cell preparation procedure.

Large fields of view, without any stitching and thus without any compromise to the high image quality achieved by the Livecyte system, delivers the ability to produce both statistically significant population analysis and the capacity to interrogate behaviour to a single cell level. This provides a far greater suite of information regarding the behaviour of the cell culture in reaction to the introduction of a particular drug.