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Proliferation Assays for Live-Cell Analysis

What is Cell Proliferation?

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.

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Incucyte® Proliferation Assays

Introducing Incucyte® Proliferation Assays

The Incucyte® Live-Cell Analysis System enables real-time, automated cell proliferation assays inside your tissue culture incubator:

(1) Label-free, Confluence

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

Monitor proliferation of A549 cells in real time with confluence image mask (Gold) using Incucyte® Proliferation Assays.

(2) Label-free, direct cell count

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.

Proliferation Cell-by-cell Figure 1

Click image to enlarge

Monitor proliferation of A549 cell in real time with Cell by Cell Analysis Software Module (mask shown in yellow).

(3) Fluorescent Labeling, Direct Cell Count

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

Monitor proliferation of HT1080 in real time using Incucyte® Nuclight Green labeled cells and cell proliferation assays.

Incucyte Proliferation Assay Concept

  1. Measure cell proliferation using live cell time-lapse imaging, with or without labels. Easily generate long-term growth and growth-inhibition curves and monitor morphology
  2. Directly measure changes in true cell number (nuclear count) over time using Incucyte Nuclight live cell labeling reagents. Automatically analyze growth curves and extrapolate doubling times.
  3. Explore co-cultures using both green and red Incucyte Nuclight Lentivirus Reagents.
  4. Multiplex proliferation readouts with measurements of apoptosis or cytotoxicity by combining with the Incucyte Caspase-3/7 Reagents, Incucyte® Annexin V Reagents or Incucyte® Cytotox Reagents

Key Advantages

Key Advantages of Incucyte® Proliferation Assays

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Visualize and quantify cell proliferation using time-lapse imaging

  • Observe cells growing over time and measure cell proliferation using intuitive Incucyte® image analysis tools and cell proliferation assays. Validate treatment effects with images and movies

Proliferation Cell-by-cell Figure 2

Confluence Phase Count Fluorescence count
Mono-culture label-free yes yes no
Mono-culture + label yes yes yes
Co-culture label no yes yes
Multiplex with cell health no yes yes

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.

Automatically analyze growth curves and extrapolate doubling times

  • Determine how and when treatment effects occurred without removing cells from the stable environment of the incubator - ideal for long-term studies (0 to >10 days)

Measure treatment effects automatically and non-invasively

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).

Simple, flexible 96/384-well protocols: no washing, no fixing, no lifting

  • Plate cells, add treatments and read kinetically in the Incucyte Live-Cell Analysis System. Read up to 6 x 384-well plates at once for medium/high-throughput screening

View the IncuCyte cell proliferation assay protocol

View the Incucyte Proliferation Assay protocol

View the labeled cell count proliferation protocol

Multiplex with apoptosis and cytotoxicity measurements

  • Combine Incucyte® Proliferation Assays with Incucyte® Cytotox, Incucyte® Annexin V or Incucyte® Caspase 3/7 Reagents for multiplexed measurements of cytotoxicity/apoptosis. Readily discriminate between cytotoxic and cytostatic treatment effects.

Proliferation Cell-by-Cell Figure 3a

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).

Proliferation Cell-by-Cell Figure 3b

Quantify time-courses and concentration-dependence of proliferation and apoptosis. Effects of camptothecin on HT-1080 cells: (A) cell count (B) apoptotic index and (C) concentration-response curves.

Explore co-cultures using Incucyte® Nuclight Lentivirus Reagents

  • Identify and quantify cells in co-culture by labeling with Incucyte® Nuclight Lentivirus Reagents

IncuCyte Co-culture cell proliferation assays

Incucyte® Co-culture cell proliferation assays.
Time-lapse movie of co-cultured nuclear-labeled HT1080 cells (green nuclei, Nuclight Green) and A549 cells (red nuclei, Nuclight Red).

Micro-environment effects on chemotherapeutic drugs

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).

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Cell Proliferation FAQs

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

Ordering Info

Ordering Information

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.

Product Qty. Catalog No.
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

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