Cell proliferation, cell death, and apoptosis assays are cornerstones of cancer therapeutic, developmental biology, and drug safety research. Correspondingly, there are many in vitro assay methodologies for performing these assays. It has been well documented, however, that existing assays exhibit drug concentration and exposure dependencies that are difficult to interpret using single time point measurement protocols.
Surprisingly, to date there is no easy-to-use, industrialized method for quantifying the number of cells and the number of apoptotic cells (or dying cells) in the same culture continuously over time. Moreover, the growing awareness of the complexity of the tumor microenvironment and the importance of cell-to-cell signaling in cancer biology support the need to measure these parameters using more sophisticated in vitro models in which multiple cells types are present.
Biochemical techniques for measuring cell proliferation responsive to LDH, ATP, or MTS reduction are generally homogeneous plate-reader assays. While simple, these assays are limited to quantifying a bulk response, typically from a monoculture, at a single time point.
High-content imaging techniques involving immunocytochemistry and/or chemical probes can measure multiple parameters on multiple cell types. However, the labeling and read-out methods employed in such systems are quite often invasive, especially when applied to long-term, repetitive, time-lapse measurements on the same cell population.
Essen BioScience’s IncuCyte ZOOM™ (live cell imaging in your incubator) and CellPlayer™ reagents provide an easy-to-use solution to these challenges. This solution is defined by the acquisition, analysis, and quantification of images from living cells that remain unperturbed by the detection method, allowing for repeated measures of cell biology over long periods of time, from days to weeks. We refer to this solution as Live Content Imaging.
Nonperturbing, Real-Time Labeling Reagents
Historically, automated time-lapse analysis of cell proliferation has been limited to nonlabeled surrogate measurements using image-based confluence or electrical impedance measurements of cell layers. While appropriate for some applications, these methods are susceptible to artifacts and nonlinearities introduced by time-dependent cell morphology changes. To address this, Essen BioScience has released a series of novel, nonperturbing lentiviral constructs designed to label the nucleus of cells with either GFP or RFP variants.
CellPlayer NucLight™ Green and NucLight Red incorporate an EF-1α-promoter with an IRES puromycin resistance cassette enabling transient and stable infection of mammalian cells. Quality control testing on primary, immortalized, and tumor-derived cells has revealed no adverse effects from lentivirus infection or nuclear fluorescent protein expression as confirmed by long term time-lapse proliferation, migration, differentiation, morphology and cell death measurements.
Traditional measurements of apoptosis are hindered by the need to determine a single measurement endpoint a priori. This becomes especially difficult when the pharmacological response is exposure and concentration dependent. Labeling protocols for apoptosis that require wash steps add a further complexity in that dying cells and cell fragments are susceptible to removal before detection.
To this end, Essen offers a nonperturbing, no-wash, kinetic, CellPlayer caspase-3/7 reagent for monitoring apoptosis in real time. This nonfluorescent, cell-permeable reagent generates a green fluorescent DNA dye signal upon cleavage by caspase 3/7 during apoptosis. Since caspase 3/7 enzyme activation is a common point of convergence, this approach measures the majority of apoptotic pathway signaling.
Case 1: Real-Time Cell Counting in Mixed Cultures
Cell-based models composed of more than one cell type in the same culture are increasingly recognized as more biologically relevant than monocultures. For example, a recent cancer study illustrated that some stromal cell types confer resistance to tumor cells in co-culture, proposing this as a possible mechanism of tumor resistance in the clinic.
Figure 1 demonstrates an approach for simultaneously measuring in real-time the proliferation of two cell types in mixed culture using the combination of CellPlayer reagents and IncuCyte ZOOM. In this simple example, two tumor-derived cell lines, A549 lung epithelial cells and HT-1080 fibrosarcoma cells, were independently labeled with NucLight Red and NucLight Green, respectively. The resulting populations were placed in co-culture and imaged at two-hour intervals over three days in IncuCyte ZOOM.
Integrated image processing algorithms provided an independent nuclear count of both cell types continuously in time. The combined attributes of this approach can be used to better elucidate the mechanism and timing of drug responses on cell proliferation in biologically relevant, mixed culture systems.
Case 2: Multiplexed Kinetic Measurements of Proliferation and Apoptosis
Experiments designed to measure the effects of genetic manipulation or compound treatment on cell proliferation can be complicated. It is often difficult to discern whether drug treatment has induced cell death (apoptosis and/or necrosis), inhibited cell growth, or a combination of the two. Using Essen BioScience’s IncuCyte ZOOM and CellPlayer reagents, researchers can now monitor cell proliferation in addition to apoptosis or cytotoxicity on the same cells over time. Figure 2 illustrates one such example.
In this experiment, MDA-MB-231 cells expressing NucLight Red are cultured in the presence of increasing concentrations of staurosporine, a potent inducer of caspase-mediated apoptosis, and are imaged every three hours in IncuCyte ZOOM.
As shown in the associated time curves, the nuclear marker provides a reliable intact nuclear count (red graph), whereas the green channel provides a means to quantify caspase-3/7 activity (green graph). Both signals exhibit a concentration and time-dependent response.
This multiplexed data can then be used to derive more informed pharmacology using any single time point or combination of time points over the duration of the experiment.
The combination of Essen BioScience’s IncuCyte ZOOM live-cell imaging system and CellPlayer reagents provides an easy-to-use, multiplexed solution for performing quantitative cell biology in real time. While this note has focused on proliferation and apoptosis, the Live Content Imaging paradigm using IncuCyte extends to a wide range of other phenotypic assays, including cell death, angiogenesis, neurite dynamics, and cell migration and invasion.