Incucyte® Cell Cycle Assays for Live-Cell Analysis
The cell cycle is a series of growth and development steps which a cell undergoes during cell division. Comprised of distinct phases, the cell cycle is a key drug discovery target and of significant importance for cancer and regenerative medicine research. A dis-regulation of the cell cycle is a hallmark of cancer, and check point inhibitors and cyclin-dependent kinases that regulate cell cycle are studied as strategies or targets for anti-cancer therapies. Cell cycle regulation is also believed to play a key role in the cell differentiation required to deliver on the promise of regenerative medicine.
However, conventional cell cycle study methods may be challenging due to:
- Capability to take only a single time-point - or concatenated time-points - from only a small number of cells or from cells that are fixed or exhausted during the experiment
- Inability to study the cell cycle over multiple cell divisions within the same cell
- Combination of independent, non-integrated solutions for cell cycle labeling, data acquisition and analysis
- Complex data analysis for evaluation of stages of the cell cycle
- Limitations on variables or number of compounds to be studied due to lack of instrument throughput
Continuous imaging and analysis over time at microplate throughput - within a physiologically relevant environment inside your incubator - enables the long-term, kinetic analysis of cell cycle dynamics. With the quantification of cell cycle progression within living cells, researchers can:
- Study cell cycle dynamics continuously over time
- Track individually identified phases of the cell cycle in individual cells
- Reveal cell cycle progression within a physiologically relevant context
- Gain deeper insight into drug-induced effects on cell cycle regulation
With the Incucyte® Cell Cycle Assay, you can now quantify cell cycle progression continuously over multiple cell divisions - inside your incubator. Gain deeper insight into treatment effects on cell cycle dynamics, or link cell cycle arrest to morphology and function using our unique and accessible approach to live-cell imaging and analysis.
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Solutions for Cell Cycle
Incucyte® Cell Cycle Assay Overview
Incucyte® Cell Cycle Assay combines easy-to-learn automated image acquisition and integrated analysis of 96- or 384-well plates with a simple, non-perturbing protocol for cell cycle labeling. The result is kinetic, image-based measurements of cell cycle progression over time – all from inside your incubator. Incucyte® Cell Cycle Lentivirus Reagents are fluorescent ubiquitination-based cell cycle indicator (FUCCI) that provide homogeneous expression of two fluorescent proteins to distinguish between cells in the interphase or the mitotic phase to enable the visualization of cell cycle progression. Incucyte® Cell Cycle Green/Red Lentivirus and Incucyte® Cell Cycle Green/Orange Lentivirus are both available for use in the Cell Cycle Assay. Used in combination with the Incucyte® Cell-by-Cell Analysis Software Module, the Cell Cycle Assay offers a powerful solution for tracking subpopulations of cells within each stage of the cell cycle over multiple cell divisions, enabling greater insight into cell cycle dynamics.
Incucyte® Cell Cycle Lentivirus Reagents enable efficient, non-perturbing and homogeneous labeling of living mammalian cells for in vitro analysis of cell cycle progression.
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Incucyte® Cell-by-Cell Analysis Software Module
The Incucyte® Cell-by-Cell Analysis Software Module enables a range of applications for mixed cultures of adherent and non-adherent cells. Perform label-free cell counts on adherent and non-adherent cells and subsequent cell-by-cell classification based on shape, size or fluorescence intensity to quantify dynamic changes in subpopulations within a mixed culture, opening a new world of discovery.
Cat. No. 9600-0031
Cell Cycle Assays with the Incucyte® Live-Cell Analysis System
The Incucyte® Live-Cell Analysis System provides researchers with real-time image acquisition and analysis and - alongside Incucyte® Cell Cycle Lentivirus Reagents - builds a quantitative picture of dynamic cellular changes. Combining the cell cycle data with cell morphology, function and cell health readouts enable multiple aspects of a treatment or culture conditions to be considered in a single well. More advanced cell models, such as co-cultures with immune cells, can also benefit from the information yielded by Incucyte® Live-Cell Analysis Systems and reagents - under physiologically relevant conditions - to deepen our understanding of temporal patterns of cell cycle progression.
Key Advantages of Incucyte® Cell Cycle Assay
Get the answers you need by monitoring cell cycle progression continuously over multiple cell divisions
A simple live-cell labeling protocol, automated acquisition and analysis in 96- or 384-well plates with easy-to-use, purpose-built software tools to reveal real-time kinetic cell cycle changes
See example data below
Track effects of drug treatments on cell cycle phases within subpopulations with the Incucyte® Cell-by-Cell Analysis Software Module
See example data below
Gather more data by multiplexing with other fluorescent reagents
See example data below
Example Data for Cell Cycle Assay
Quantify cell cycle dynamics
Follow cell cycle progression over multiple cell divisions in a variety of cell types using image-based measurements. Maintain physiological relevance with stable incubation provided by your incubator and our novel Incucyte® Cell Cycle Lentivirus Reagents that are non-perturbing to cell health and morphology.
Figure 1. Generate image-based measurements of cell cycle transitions over multiple cell divisions with automated image acquisition and integrated analysis. HeLa cells transfected with the Incucyte® Cell Cycle Green/Red Lentivirus exhibit red fluorescence in G1 and green fluorescence in S/G2/M (A). Yellow cells are in transition from G1 to S while non-fluorescent cells are moving from M to G1. Quantification using Incucyte® Cell-by-Cell Analysis enables accurate masking shown by yellow mask (B) and classification based on red and green fluorescence (C&D). Panel E shows quantification of cell cycle duration, where HeLa cells transfected with Incucyte® Cell Cycle Green/Red Lentivirus were treated with Thymidine (2.5 mM for 24 h) to arrest cell growth in S/G2/M phase. Upon release of this block, cells undergo synchronous division (M-G1-S/G2/M). Data shows a cycle length of 17 h (peak to peak duration). Data shown as mean ± SEM of 6 wells.
Figure 2. Generate direct measurements within living cells through non-perturbing reagents. The introduction of the Incucyte® Cell Cycle Green/Red Lentivirus stably into cells is non-perturbing. Images show the same morphology in parent or transfected MDA-MB-231 cells and analysis of images demonstrates no effect on cell growth.
Unlock productivity
Speed time to answer with Incucyte's approachable, end-to-end solution. Follow a simple live-cell labeling protocol - no fixing, no lifting. Automate acquisition and analysis in 96- or 384-well plates with easy-to-use, purpose-built software tools. Easily generate graphs to reveal kinetic changes in cell cycle phase distribution using Incucyte® Cell-by-Cell Analysis Software Module.
Figure 3. Incucyte® Cell Cycle Lentivirus Quick Guide. Stably express a fluorescent ubiquitinated-cell cycle indicator (FUCCI) using a simple labeling protocol — no fixing, no lifting.
Gain new insight
Combine the Incucyte® Cell Cycle Lentivirus with Cell-by-Cell Analysis to track individually identified phases of the cell cycle for evaluation of drug treatment effects, or link drug-induced cell cycle arrest to cell differentiation.
Figure 4. Measure drug effects on cell cycle phase distribution over time. Time course of HT1080 fibrosarcoma cell division following cisplatin or fluorouracil (5FU) treatment. Cell cycle phases were determined in HT1080 cells expressing the Incucyte® Cell Cycle Green/Red Lentivirus. Images were analyzed using Cell-by-Cell Analysis Software and population subsets classified based on green and red fluorescence. Microplate views (top row) show the concentration and time dependent effects over 30 h of cisplatin and 5FU compared to vehicle for the G1 phase (red population), S/G2/M phase (green population) and G1-S phase (orange population). The concentration response curves taken at 24 h post treatment (bottom row) show after cisplatin treatment, there was a decrease in the population of cells in the G1 phase, increase in the S/G2/M phase and decrease in the G-S phase in line with its effect to interfere with mitosis. After 5FU treatment, there was an increase in the population of cells in the G1 phase, decrease in the S/G2/M and G1-S phase in line with effect on DNA synthesis. Values shown are the mean ± SEM of 6 wells.
Figure 5. Observe drug-induced cell cycle arrest effects on cell morphology and function. THP-1 monocytes stably expressing the Incucyte® Cell Cycle Green/Red Lentivirus were exposed to vehicle or Phorbol 12-myristate 13-acetate (PMA; 100 nM) and analyzed using the Cell-by-Cell Analysis Software. PMA causes the cells to differentiate arresting the cell cycle in the G1 phase (A). Analysis demonstrates a lack of proliferation (B), increase percentage of red cells, G1 phase and a decrease in green cells S/G2/M phase (C) compared to vehicle treated cells. PMA-treated cells were also larger in area (D), indicative of a morphological change and concordant increase in phagocytic potential as measured by efferocytosis of apoptotic Jurkat cells labeled with the pHrodo® Red Cell Labeling Kit for Incucyte® (E).
Derive deeper biological insight
Gather more data points and findings in drug screens by multiplexing with fluorescent cell health reagents to add insight into compound mechanisms.
Figure 6. AU565 cells expressing Incucyte® Cell Cycle Green/Orange were treated with carboplatin (100 μM), lapatinib (0.1 μM) and camptothecin (10 μM) in the presence of Incucyte® Annexin V NIR Dye (1:200). Scans were acquired and analyzed in Incucyte® SX5 using the Incucyte® Cell-by-Cell Analysis Software Module. Time-courses show the populations of cells in S/G2/M (green) or G1 (red) overlaid with the population of Annexin V positive objects (teal). Carboplatin arrests cells in S/G2/M. Lapatinib targets specific cell receptors and is, therefore, effective on AU565. Camptothecin induces apoptosis. Phase and fluorescence (green, orange NIR) blended images show AU565 cells at 3 days post treatment.
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Cell Cycle Frequently Asked Questions
The Incucyte® Cell Cycle Green/Red Lentivirus is compatible with the Incucyte® SX1 and S3 Live-Cell Analysis Systems. It is also compatible with the Incucyte® SX5 when configured with the Green/Red Optical Module.
The Incucyte® Cell Cycle Green/Orange Lentivirus is compatible with the Incucyte® SX5 when configured with the Green/Orange/NIR Optical Module.
The Incucyte® Cell Cycle Green/Orange Lentivirus, which is compatible with the Green/Orange/NIR Optical Module, can be multiplexed with Near-IR reagents such as the Incucyte® Annexin V NIR or Incucyte® Cytotox NIR to add insight into compound mechanisms.
Yes. This reagent is compatible with variety of adherent and non-adherent cell types, and should work in most cells.
Compatibility with cell types will be same as other lentivirus reagents, and should work for most cancer/stem cells, but perhaps not immune cells.
Yes. Lentiviral delivery of this reagent enables unlimited expression for the transfected cell line.
No. The introduction of the Incucyte® Cell Cycle Lentivirus into cells is non-perturbing, where no change is seen in cell morphology or growth.
With PI, you will be able to identify different cell cycle phases and may get more precise measurements. However, this method uses dead cells, with no ability for long-term kinetics and studying multiple cell divisions within the same cell which the Incucyte® Cell Cycle Lentivirus provides.
The Incucyte® Cell-by-Cell Software has the advantage of being able to accurately quantify all 4 phases based on the subpopulation, whereas with Basic Analyzer only 3 phase can be quantified (cannot calculate colorless population. Cell-by-Cell would also be able to provide label-free cell counts to evaluate proliferation, whereas Basic Analyzer would be looking at proliferation based on total area confluence. To evaluate cell morphology, Cell-by-Cell would provide individual cell area/eccentricity measurements, while Basic Analyzer would rely on qualitative evaluation. Finally, Cell-By-Cell provides simple to use, integrated analysis where Basic Analyzer would require data exportation for additional manipulation.
Bottom line, that while Cell-By-Cell Analysis is more robust, both Cell-by-Cell and Basic Analyzer will detect trends and mechanism of action of cell cycle.