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Monday, October 21 • 2:30pm - 3:00pm
Poster Presentation #2- Time-Lapse Imaging of Cell Behaviour on 2D Substrates and in 3D Aggregates: Watch it Live with DRAQ9

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The importance of cell migration and proliferation in wound healing, tumour metastasis and tissue remodelling is under continued investigation while the ability to track single cells and cellular communities has become a potent tool to measure their biological functional capacity and a systems response to perturbation. Delivering a 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 describe a cell permeant, far-red fluorescent, cytoplasmic probe DRAQ9 (BioStatus) that is non-toxic, and present continuously as real-time tracker so fluorescence is undiminished over time e.g. during spheroid aggregation, formation and expansion; in 2D scratch-wounds; or a hybrid assay, as single 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 cell tracking in aggregates, migration in 2D or indeed through mitosis.

Additionally, in end-point analyses (e.g. high throughput toxicity assays) a fundamental first step to 3D object image analysis is correct boundary identification, where failure results in inaccurate downstream morphometric analysis. DRAQ9 functions as a whole 3D-object “paint”. This allows accurate reproducible boundary identification, using an inexpensive universal platform, simplifying image analysis, for rapid, effective screening.

A459 cells were cultured in wells using a 2 Well insert (IBIDI). After 24 h inserts were removed, cells washed and DMEM (with 10% FBS, 2 μM DRAQ9) added then returned to the incubator for 20 min. Time-series images were acquired every 20 min. using 20x objective lens, for 24 h. Cells were manually tracked and analysed using imageJ. Spheroids were generated in a U-bottomed low-adherence plate from A549 cells cultured in DMEM (with 2 μM DRAQ9, upon spheroid culture) for 7 days. Time-series images were acquired every 30 min. using 10x objective lens, for 4 days. After 7 days spheroids were re-plated to adhere for outgrowth and time-series images acquired every 20 min. using 10x objective lens, for 48 h. A widefield microscope was used throughout.

Manual cell tracking can be completed with confidence due to DRAQ9's cytoplasmic localisation and absence in nuclei. Distinct cell patterning enables automated cell tracking algorithms. Mitosis can be visualised from changes in DRAQ9 signal due to altered morphology. Spheroid painting allows confident morphometric analysis of 3D cultures over time due to non-toxic properties of DRAQ9 for time-series analysis of cultures. Emerging cell behaviour can be visualised from 3D cultures.

DRAQ9 labeling of live cell cultures enables real-time tracking of cell migration and cell division. Visualisation of cell-cell and cell-matrix interactions provides rich information on cell behaviour within a niche. Far-red fluorescence makes it compatible with imaging on sub-optimal imaging substrates e.g. tissue culture plastic. Changes in fluorescence intensity adds information on cellular division, allowing such events to be localised to a cell 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 automated image analysis, reducing image analysis times.

Speakers
RE

Roy Edward, BSc Hons

Sales and Marketing Director, BioStatus Limited
Trained in Biochemistry (St Andrews Univ.). 30+ years of international experience in biotools, in biomagnetic separation and proteomics.Beyond the UK, worked in the USA and Norway with roles in product and marketing management, international distributor management and business development.Written... Read More →


Monday October 21, 2019 2:30pm - 3:00pm
Sherry Coutu Seminar Suite Foyer