Cell Based Assay Development – Live-cell Imaging

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Fixed time point versus kinetics: Studying cancer cell health and compound activity in a nonintrusive kinetic manner using the Incucyte SX5 imager. 

The ‘analogy of the highway.’ “I stand on a bridge over a highway with my camera facing directly down. I take images every three seconds. If I am lucky, I may see a section of a car in every third image and this may lead me to believe that the road is relatively empty. Now I look up and raise my camera along the road from the bridge and I see a different image – many cars. My perception was affected by the time points at which I took my initial images.”

And so to biology, individual time points versus kinetics data, looking down or looking out along the road.


Cell-based assays are at the cornerstone of many drug discovery programs. Understanding the optimal culture conditions for the chosen cell type is the first step in any assay optimisation process and has multiple potential benefits, including but not limited to, speeding up the assay optimisation process.

The SX5 is the first step in our assay optimisation workflow. At the early stages of assay optimisation, the benefits of simple but highly accurate confluency monitoring in real-time are of profound importance. It is widely acknowledged that the human eye is a highly subjective scorer of confluency. 

Phentoypic assay live cell imaging

Figure 1. Image of the Incucyte SX5 kinetic imager. This instrument sits inside the incubator and continuously monitors cells in well plates and flasks by taking images of cells at defined location, defined magnification, and defined time points.

The SX5 uses the phase contrast image to mask the cells and accurately determine the percentage of the surface that is occupied by cells. This approach provides a deeper understanding of the optimal assay conditions and achieves better decision making on seeding density and time points for robust and reliable cell-based assays across a broad range of cellular readouts.

Taking quantification to the next level using the Cell-by-Cell analysis algorithm, the SX5 can analyse an image and precisely count the number of cells that are, for example, stained for Annexin V or Caspase 3/7. Annexin V or Caspase 3/7 are used as markers of cell death when evaluating the activity of compounds for cancer cell biology. By using the Incucyte, we can observe the rate of cell death through the kinetic expression of Caspase and Annexin.

What We Did...

Confluency Measurements

The confluency of two cell lines with differing growth rates was measured over a 96-hour time course with data acquired every 6-hours. The instrument uses the phase image to mask the area occupied by cells to determine a percentage confluency. The Cell-by-Cell algorithm can also be used to count individual cells for more detailed analyses. The time course allows our scientist to identify an optimal seeding density, where the cells remain sub-confluent over the duration of the assay. This information feeds into the design of screening assays such as a proliferation end point assay – in this case CellTiter-Glo®2.0 after 96 hours of culture.

cell based assay development - live cell imaging

Figure 2. “The best data” are generated when your cells are in the exponential phase of growth. Confluency measurements taken from two different cell lines over a 96 hour period (left) demonstrating the differential growth rates at different seeding densities. This affects cell health models of disease and influence the toxicity of compounds using say a CellTitre Glo reagent to examine ATP generation / turnover (center). The Incucyte can drill down into the images if necessary by using the Cell-by-Cell analysis module to provide more detailed data.

Apoptosis Assay - A Route to Cell Death - Homogeneous Format and Continuous Monitoring

Apoptosis assays are widely used amongst several cell health assays. The Annexin V green reagent measures the appearance of phosphatidylserine on the outside of the cell membrane, an early sign of apoptosis. Caspase 3/7 reagents are also used to measure cells committed to apoptosis. Whilst there are other assays for measuring both parameters, the Incucyte SX5 assays benefit from the homogeneous format and continuous monitoring rather than a fixed end point assay. 

Figure 3.  Detection of Annexin V appearance on the outer surface of the cells, a indicator of cell toxicity, as shown here following treatment with a compound that induced dose-depdendent cell death over a 96 hour period in culture. 

Figure 4. Staurosporine induced rapid Caspase 3/7 activation in cells over a 24 hour treatment period. Caspase staining appears rapidly then decays beyond 24 hours as cell death occurs and this population is ignored by the algorythm.

cell based assay development - live cell imaging

Figure 5.  Phase image (left) and cell-by-cell image (right) of the same frame. In the phase image, it is possible to observe the green fluorescence, consistent with Caspase 3/7 activation, whilst in the Cell-by-Cell image, it is possible to assign Caspase activation to individual cells as this population is distinguished by the algorthym.

Real Time Labelled Assays

1. Chemotaxis of HT-1080 Cells

HT-1080-Red cells were added to the upper chamber of the Incucyte® Clearview 96-well plate for Chemotaxis measurement. Varying percentages of serum in media were added to the lower chamber. Cells migrated through the pores towards the chemoattractant and were imaged by the SX5 in real time. It is also possible to count both cells that have migrated and those that have not to obtain a migration ratio.

cell based assay development - live cell imaging

Figure 6. HT-1080 cells migrate towards a gradient of serum over a period of 50 hours in culture (left). Time lapse images of cells that have migrated from the upper to the lower chamber and are labelled with a red mask by the Incucyte algorthym (right)

2. GFP Transfection in HEK293 Cells

The efficiency of transfection was monitored in HEK293 cells transfected with a GFP-tagged target protein. Multiple transfection conditions were investigated simultaneously. Images were masked in pink for cells with fluorescence greater than the background. 

Figure 7. Transfection efficiency as monitored by the appearance of a GFP-tagged protein construct transiently transfected into cells.

3. Spheroid Assays

HT-1080-Red cells were cultured at 3 different densities to form spheroids in 96 well S-Bio U bottom plates. Cells were imaged over 6 days in the brightfield and red channel to generate time lapse movies. Masks were generated using the spheroid brightfield image to measure spheroid size over time. Spheroid health was also monitored by measuring the red integrated intensity over time. As the spheroid became more compact over time and developed a necrotic centre the red intensity decreased.

Figure 8. Spheroid health over time as monitored by spheroid size and red intensity, a marker of healthy cell number.

cell based assay development - live cell imaging

Figure 9. Kinetic images of spheroid health over a 6-day incubation period as determined by brightfield object area. Inclusion of compounds that result in toxicity decrease either spheroid size (by compaction) or the numbers of cells (a decrease in fluorescence intensity).


This case study highlights the use of the Incucyte SX5, its algorithms and specialist plates, to study cell confluency, cell health, cell migration, transfection efficiency, spheroid health and size. A number of these parameters are used by Charnwood Discovery in our projects either in the oncology therapeutic area or the inflammatory therapeutics area. The study of biological mechanisms and how these are affected by compound treatment is very important from a kinetic viewpoint.