Page 6 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

While traditional compact microscopes typically only image from The real-time live-cell analysis approach also provides the
a single micro-plate or flask at a time, new live-cell analysis opportunity to make data driven decisions while the experiment
devices such as IncuCyte can automatically capture and analyze is in progress. A researcher studying the biology of vascular
images from multiple micro-plates in parallel, thereby significantly or neuronal networks, for example, may wish to first establish
increasing throughput (e.g. IncuCyte = 6 x 384 well plates). With a stable network before assessing the effects of compound
the IncuCyte® system, a unique moving optical path design means treatments or genetic manipulations (e.g. siRNAs). With
that the cells and cell plates remain stationary throughout the continuous live-cell analysis, it is straightforward to temporally
entire experiment. This further minimizes cell perturbance and track network parameters and use the real time data to judge
enables imaging and analyses of both adherent and non-adherent when best to initiate the treatment regimes. The timing of
cell types. adjunct studies such as analysis of metabolites or secreted
proteins in supernatants can also be guided. Drug washout
This combination of functionality, throughput and ease of use studies may be performed using the real time data to identify
revolutionizes the way researchers can think about imaging when an equilibrium response occurs and to trigger the timing
assays in living cells. Real time live-cell analysis has now been of the washout regime. If for any reason it transpires that the
applied to a wide range of phenotypic cellular assays including experiment is not performing as expected then treatments
cell proliferation, cell death and apoptosis, immune-cell killing, could be withheld to save expensive reagents and follow-on
migration, chemotaxis, angiogenesis, neurite outgrowth and experiments can be initiated more quickly to make up time.
phagocytosis. In each case, the full time-course data and ‘mini-
movies’ of the assay provide greater biological insight than end Real-time live-cell analysis is extremely helpful when developing,
point assays. Novel analyses such as area under curve, time to validating and troubleshooting phenotypic assays. Within a small
signal onset or threshold, and rate parameters (dx/dt) are at times number of assay plates it’s usually possible to obtain a clear
highly value adding. Simply calculating the assay signal at its peak understanding of the relationship over time between assay signal
timepoint and/or at the optimal signal/background all helps in and treatments, cell plating densities, plate coatings and other
assembling robust and reproducible assays. Of course, transient protocol parameters. Scrutiny of the kinetic data and ‘mini-movies’
effects of treatments can be detected by kinetic imaging that from each well help to rapidly pinpoint sources of within- and
may otherwise be missed with end-point reads. across-plate variance and to validate the biology of interest.
This is particularly true for more advanced cell systems such as
Due to its non-invasive nature, measurements from cells can be co-cultures where far more permutations and combinations of
made not only during the assay itself but also during the cell protocol parameters exist (e.g. cell plating ratios) and the biology is
preparation and ‘pre-assay’ stage. For example, the morphology more complex.
and proliferation rates of cells can be monitored throughout the
cell culture period and immediately post-seeding on the micro- In summary, real-time live-cell analysis is re-defining the
titer assay plate. The parameter/phenotype of interest can be possibilities and workflows of cell biology. The combination of
measured prior to the addition of treatments to provide a within ease of use, throughput, long term stability and non-invasive
well baseline measure. Quality control of cells and assay plates measurement enables researchers to monitor and measure
in this way helps improve assay performance and consistency by cell behaviors at a scale and in ways that were previously not
ensuring that experiments are only conducted on healthy, evenly possible, or at the least, highly impractical. In the following
plated cultures with the expected cell morphology. chapters of this handbook, we illustrate this with a range of
different application examples.

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