Cell proliferation is the biological process of cells increasing in number over time through cell division ('mitosis' in eukaryotic cells). Proliferation is an essential mechanism for normal tissue development, regeneration and renewal. Aberrations in cell proliferation, however, can give rise to malignant transformation and cancer pathology.
Cell proliferation assays are a cornerstone of stem cell and cancer cell pathway analysis and drug testing for efficacy and safety. There are three main types of biochemical cell proliferation assays, based on DNA synthesis (e.g. 3H thymidine incorporation, BrdU), metabolic activity (e.g. Alamar blue, LDH) and ATP concentration (luciferase bioluminescence). Whilst each have their merits, the majority are single end points or at best a series of concatenated endpoints to measure the time-course. Most are indirect and subject to artifacts that cannot be readily verified by morphology changes.
The Incucyte® Live-Cell Analysis System enables real-time, automated cell proliferation assays inside your tissue culture incubator:
Cell proliferation is monitored by analyzing the occupied area (% confluence) of cell images over time. As cells proliferate, the confluence increases. Confluence is an excellent surrogate for proliferation, until the point that cells are densely packed or when large changes in morphology occur.
Monitor proliferation of A549 cells in real time with confluence image mask (Gold) using Incucyte® Proliferation Assays.
Using the Incucyte® Cell-by-Cell Analysis Software Module, cell proliferation can be quantified by counting the number of phase objects over time. By identifying individual cells, a label free count can be achieved until the point that cultures become densely packed and cell edges cannot be accurately determined. In addition, subsequent classification of cells into subpopulations can be performed based on properties such as size, shape and fluorescence intensity.
Monitor proliferation of A549 cell in real time with Cell by Cell Analysis Software Module (mask shown in yellow).
Cell proliferation is quantified by counting the number of fluorescent nuclei over time to give true cell growth rates. Cells are labeled with nuclear-restricted non-perturbing fluorescent labels (e.g. Incucyte® Nuclight Green Lentivirus Reagent). In co-culture, different labels can be combined to simultaneously measure proliferation of two cell types.
Monitor proliferation of HT1080 in real time using Incucyte® Nuclight Green labeled cells and cell proliferation assays.
|Confluence||Phase Count||Fluorescence count|
|Mono-culture + label|
|Multiplex with cell health|
Quantify cell proliferation in real-time using cell confluence (second column), Cell-by-Cell Analysis (third column) or fluorescence cell counting (forth column) in HT-1080 fibrosarcoma cells. For fluorescent cell counting, cells were labelled with the non-perturbing nuclear label, Incucyte Nuclight Red Lentivirus Reagent. Note the increase in confluence or object number over time and corresponding metrics. Table compares the use of the various approaches.
Measure treatment effects automatically and non-invasively. Incucyte Proliferation Assays allow every well of a 96/384 well plate to be imaged and analyzed automatically to provide a microplate readout of cell proliferation over time (A). Proliferation time-courses reveal concentration-dependent treatment effects (B). Transform data into concentration-response curves to compare pharmacology (C).
Multiplex live-cell counting with apoptosis measurements using Incucyte Proliferation Assays. Camptothecin (1 µM) treated HT-1080 fibrosarcoma cells in the presence of the Incucyte Caspase 3/7 reagent to detect apoptosis. Using Incucyte Cell-by-Cell Analysis it is possible to identify individual cells within the population and classify them based on green fluorescence enabling the calculation of an apoptotic Index (% positive green cells within population).
Micro-environment effects on chemotherapeutic drugs. The role of stromal cells in breast cancer cell resistance to Lapatinib. SK-BR-3 breast adenocarcinoma cells labeled with the Incucyte® Nuclight Red Lentivirus Reagent were grown in monoculture or in co-culture with unlabeled stromal fibroblasts. Incucyte live-cell imaging and analysis revealed that Lapatinib potently inhibits SK-BR-3 cell growth in monoculture (left) but the potency is reduced if SK-BR-3 cells are grown in co-culture with stromal fibroblasts (center and right).
Tracking cell growth, or cell proliferation, in cultures is relatively easy, if you know what to look for. The simplest method of monitoring cell growth is to track a culture’s progression toward confluence. Confluence describes the percentage of the well or plate that is covered in cells. Confluence is a useful metric in the everyday maintenance of cell lines and cell-based assays, like transfection. However, confluence is not a useful metric when absolute cell numbers are needed. In those cases, cell proliferation may be tracked either label free using the IncuCyte Cell-by-Cell Analysis Software or with the use of a nuclear-labelling reagent. Each nucleus imaged would equate to one cell. Serial measurement of both confluence and cell number are possible with a live-cell analysis system.
Regarding multinucleate cells, counting nuclei would not be an appropriate measure of cell number. However, a cell proliferation assay’s readout of cell confluence would be capable of reporting an increase in the total biomass of the cell population – and this readout may be sufficient for applications where cell density/confluence are important, like transfection.
DNA synthesis-based analysis of cell proliferation involves the use of artificial DNA bases during DNA replication, leading to the incorporation of the artificial bases in the newly formed double helix. These bases are then stained with anti-BrdU antibodies, providing a rough idea of the number of cell divisions that took place during the BrdU incubation. Analysis of BrdU staining requires the fixation of the cells, making them unsuitable for downstream use.
Live-cell analysis provides deeper insight into the process of cell proliferation, enabling the monitoring of proliferation rate and cell morphology while maintaining a constant cell-growth environment. Additionally, the IncuCyte™ cell proliferation assay doesn’t require the use of any dyes or labels for confluence measurement, making the cells suitable for use in downstream assays.
Monitoring cell proliferation in co-cultures requires non-perturbing labels (that will not interfere with the cells’ normal division process) and a method for monitoring the two fluorochromes. The process is simple and straightforward using IncuCyte’s NucLight™ red and green reagents for nuclear labeling. The two populations are transduced with either the red or green reagent and then seeded for co-culture. Cell proliferation is then tracked for each cell type. This type of live-cell analysis offers the obvious advantage of documenting the process of proliferation in the context of a co-culture, while tracking a single (or several plates of) cells across their entire growth phase. With the introduction of IncuCyte Cell-by-Cell Analysis Software it is possible to identify you cells in the co-culture by various methods e.g. labelling of surface epitopes with AB. Only one cell type needs to be labelled in the co-culture then using the classification tools sub populations can be identified in the total population (classified by mean intensity, size or shape).
Yes, the interaction of cells in complex, tissue-like relationships can be studied in vitro with co-cultures, and cell death can be tracked in those co-cultures with markers of apoptosis and membrane integrity. For the most meaningful data, cell proliferation and cell death can be monitored at the same time in the same culture, with live-cell imaging.
To track a specific cell type throughout the co-culture experiment, start by transducing the cells with one of the NucLight™ reagents, ensuring that it is a different color than the Cytotox reagent used. The cells will grow and associate into dense, interacting cultures, and cell death will be monitored with the Cytotox or Caspase 3/7 reagents.
Note: All dead and dying cells will be positive for the Cytotox or Caspase 3/7 reagent, not just the ones transduced with the NucLight reagent.
The metrics of confluence and cell number are essential for certain laboratory applications involving normal, healthy cells. Cells that are clumped and form thick multilayer lawns are not good candidates for assessing confluence or cell number with optical techniques. Confluence assessment assumes that cells are growing in a monolayer, only contacting other cells in two dimensions. Determining cell count with cell proliferation assays requires a nuclear staining reagent, and non-monolayer cells may have inadequate contact with the reagent, leading to suboptimal uptake across the population. Cells obscured by overlayered cells may not be correctly visualized by the optics, leading to blurry or absent signal. For optimal results, cell proliferation assays should be conducted with cell monolayers.
The morphological change from a healthy nucleus to an apoptotic nucleus is striking, and easy to detect visually. When cells enter apoptosis, the nucleus begins to condense, becoming smaller and darker. Eventually, the nucleus fragments (karryohexis). During a cell proliferation assay, you may be able to set a gate for live-cell nuclei vs. apoptotic-cell nuclei. Additionally, if you’re using a visual tracking system for live-cell analysis, you could multiplex with a cytotoxicity assay or an apoptosis assay, that can let you know if your cells’ membranes are compromised or they have begun an apoptotic cascade
Learn more about cytotoxicity assays
Learn more about apoptosis assays
The Incucyte® Nuclight Reagents have been validated for use with the Incucyte® Live-Cell Analysis System. They can be combined with the Incucyte® Caspase 3/7 Reagent, Incucyte Annexin V Reagents or Incucyte® Cytotox Reagents for multiplexed measurements of apoptosis and cytotoxicity in the same well.
|Incucyte® Caspase-3/7 Green Reagent||20 μL||4440|
|Incucyte® Caspase-3/7 Red Reagent||20 μL||4704|
|Incucyte® Caspase-3/7 for Metabolism||20 μL||4776|
|Incucyte® Annexin V Green Reagent||1 vial||4642|
|Incucyte® Annexin V Red Reagent||1 vial||4641|
|Incucyte® Annexin V Orange Reagent||1 vial||4759|
|Incucyte® Annexin V NIR Reagent||1 vial||4768|
|Incucyte® Cytotox Red Reagent||5 μL x 5||4632|
|Incucyte® Cytotox Green Reagent||5 μL x 5||4633|
|Incucyte® Cytotox NIR Dye||100 μl||4846|
|Incucyte® Nuclight Green Lentivirus (EF-1α, Bleo)||0.2 µL||4626|
|Incucyte® Nuclight Green Lentivirus (EF-1α, Bleo)||0.6 µL||4477|
|Incucyte® Nuclight Red Lentivirus (EF-1α, Bleo)||0.2 µL||4627|
|Incucyte® Nuclight Red Lentivirus (EF-1α, Bleo)||0.6 µL||4478|
|Incucyte® Nuclight Green Lentivirus (EF-1α, puro)||0.2 µL||4624|
|Incucyte® Nuclight Green Lentivirus (EF-1α, puro)||0.6 µL||4475|
|Incucyte® Nuclight Red Lentivirus (EF-1α, puro)||0.2 µL||4625|
|Incucyte® Nuclight Red Lentivirus (EF-1α, puro)||0.6 µL||4476|
|Incucyte® Nuclight Orange Lentivirus (puro)||0.2 µL||4771|
|Incucyte® Nuclight NIR Lentivirus (puro)||0.2 µL||4805|
|Incucyte® Cell Cycle Green/Orange Lentivirus (puro)||0.2 µL||4809|
|Incucyte® Cell Cycle Green/Red Lentivirus (puro)||0.2 µL||4779|
|Incucyte® Nuclight Rapid Red Reagent||50 µL||4717|
|Incucyte® Nuclight Rapid NIR Reagent||50 µL||4804|
|Incucyte® Cell-by-Cell Analysis Software Module||1 module||9600-0031|