Abstract

The importance of cell migration and proliferation in wound healing, tumour metastasis and tissue remodelling is clear yet delivering real-time tracking capability and robust assay readouts is multi-faceted. It requires a cell label that is non-toxic, stable throughout, as cell identifier/locator. We utilise a cell permeant, far-red fluorescent, cytoplasmic probe DRAQ9 that is non-toxic and present continuously as real-time tracker: fluorescence is undiminished over time e.g. in spheroid aggregation, formation, expansion; 2D scratch-wounds; or hybrid assay, as cells emerge onto 2D substrate from a spheroid. In all cases, DRAQ9 labels cells for unique identification over time. This stable tracker provides a readout ripe for automated tracking in aggregates, migration in 2D or through mitosis.

Additionally, in end-point analyses (e.g. toxicity assays) the first step to 3D object image analysis is correct boundary identification. DRAQ9 functions as a whole 3D-object “paint” for accurate boundary identification, by widefield microscopy, simplifying analysis for rapid, effective screening.

We will show manual cell tracking can be confidently completed by DRAQ9's cytoplasmic localisation and absence in nuclei. Distinct cell patterning enables automated cell tracking algorithms. Spheroid painting allows confident morphometric analysis of 3D cultures over time due to non-toxic properties of DRAQ9 for time-series analysis. Emerging cell behaviour can be visualised from 3D cultures.

DRAQ9 labeling of live cell cultures enables real-time tracking of cell migration and division. Far-red fluorescence is compatible with imaging on sub-optimal substrates e.g. plastic. Changes in fluorescence intensity adds information on cellular division, allowing localisation to a niche. DRAQ9-brightfield image overlays make manual tracking easier to perform, increasing confidence in cell identification, especially during cell division. Importantly, absence of DRAQ9 in nuclei provides a mask for rapid automated image analysis.