Incucyte®️ Proliferation Assays for Live-Cell Analysis
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. Detecting changes in proliferation, as well as the ability of cells to divide in response to stimulus, is critical in oncology and stem cell research as well as drug testing for efficacy and safety.
Although there are multiple types of biochemical cell proliferation assays, challenges exist that limit a scalable method for robust quantification:
The Incucyte® Live-Cell Analysis System alleviates these shortcomings by using multiple strategies to measure the same sample over time. Using either integrated, powerful software tools and artificial intelligence (AI), or combining this label-free approach with non-perturbing reagents, quantification of cell proliferation at a microplate level has never been easier.
Incucyte® live-cell analysis solutions include:
Incucyte® Proliferation Assay Concept
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The Incucyte® Live-Cell Analysis System enables real-time, automated quantification of cell proliferation assays inside your tissue culture incubator:
Confluence can now be measured in two different ways - both included in the Incucyte® Base Analysis Software. The first method, Classic Confluence, is the existing segmentation algorithm that allows the user to tailor phase segmentation parameters to individual cell types and experimental conditions. The second segmentation method, the new AI Confluence algorithm, is a robust, AI-driven analysis designed to adapt to a wide range of cell types and morphologies while requiring less user input.
Monitor proliferation of SKOV3 cells in real time with confluence image mask (Yellow) using Incucyte® Base Analysis Software with AI Confluence segmentation.
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. In addition, subsequent classification of cells into subpopulations can be performed based on properties such as size, shape and fluorescence intensity.
The Incucyte® Advanced Label-Free Classification Analysis Software Module is available as an add-on that allows objective tracking of adherent cell populations kinetically.
Monitor proliferation of NALM6 cells in real time with Incucyte® 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). In co-culture, different labels can be combined to simultaneously measure proliferation of up to three cell types.
Monitor proliferation of HT-1080 cells stably expressing Incucyte® Nuclight Green, Nuclight Orange or Nuclight NIR Lentivirus in real time using Incucyte® Cell-by-Cell Analysis Software Module.
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Quantify changes in proliferation using label-free, intuitive software analysis.
The Incucyte® Base Analysis Software includes two different tools to measure confluence: the AI Confluence Analysis, and the Classic Confluence Analysis. Fluorescence image acquisition and analysis of Cell Health or Viability assays are also available with Incucyte® Base Analysis Software, Classic Confluence Analysis definitions.
The AI Confluence Analysis is driven by a neural network trained on many classes of cells, which provides a simple, objective, and repeatable workflow with minimal user input for highly accurate segmentation of cells in phase contrast images.
The Classic Confluence Analysis enables users to refine phase segmentation parameters to individual cell types and experimental conditions to accurately distinguish between background.
Figure 1: Analyze label-free confluence with integrated software tools. AI-driven confluence analysis offers efficient, objective analysis (A workflow). Classic Confluence Analysis utilizes incorporates user-defined segmentation based on experimental conditions (B workflow).
Figure 2. Robust segmentation of cell confluence using AI Confluence Analysis. A549, MDA-MB-231, SKOV3 and HT-1080 cells were seeded into a 96-well plate at a range of densities (0.5 – 10K cells/well). High definition (HD) phase-contrast images were acquired every 2h and cell proliferation was measured over 4 days. Yellow outline indicates AI Confluence segmentation and plate view reports % Confluence over 4 days.
Real-time measurements of cell health and proliferation are essential for studying the effects of drugs, culture conditions or genetic modifications on cell growth or viability. Identify individual cells and generate label-free, true cell counting, in both non-adherent or adherent cell modules, with the Morphological Analysis in the Incucyte® Cell-by-Cell Analysis Software Module.
Figure 3 : Label-free cell counting in adherent and non-adherent cell types using Incucyte® Cell-by-Cell Analysis Software Module. Various densities of adherent Incucyte® Nuclight Red A549 or non-adherent Incucyte® Nuclight Red Jurkat cells were analyzed over time with Incucyte® Cell-by-Cell Analysis Software Module. Red Object Count was used to validate the Incucyte®’s label-free cell counting method. Images demonstrate individual cell masking using the Cell-by-Cell Analysis. Time course of Phase Count and Nuclight Red Count across densities shows overlay of label-free and fluorescent data. This validation has been repeated across a range of cell types (date not shown). Values shown are mean ± SEM for 4 wells.
Table 1. Drugs identified in literature as relevant to cell proliferation. To assess many pharmacological agents simultaneously, 16 literature-standard compounds (Table 1) were applied to HT-1080 cells in a 384-well format.
Figure 4. 384-well microplate view of Incucyte® Nuclight Green HT-1080 cell proliferation in response to treatment. Incucyte® Nuclight Green HT-1080 cells were treated with16 different compounds, 11-point concentration-response curves in duplicate (different colors, high to low concentrations left to right). Columns 15 and 16 are vehicle (0.5% DMSO) and CHX (3 μM) controls, respectively. Note the potent concentration-dependent inhibition of cell proliferation for certain compounds (e.g., Row J, Row M, Row O), and weaker effects/inactivity of others (e.g., Row A, Row P). Abscissa: Time (0-72 hours), ordinate: Fluorescent Object Count per well (0-3800).
Combine Incucyte® Proliferation Assays with Incucyte® Cytotox, Incucyte® Annexin V or Incucyte® Caspase 3/7 Dyes for multiplexed measurements of cytotoxicity/ apoptosis. Readily discriminate between cytotoxic and cytostatic treatment effects
Confirmation of apoptotic cell death with two readouts (Caspase-3/7 and Annexin V) on the Incucyte® live-cell analysis system. Note: data is shown as example and is not related to studies mentioned in this interview.
Figure 5: Detect both Caspase 3/7 and Annexin V signals in cultures. Automatically determine the time courses of apoptotic cell death and correlate with label-free confluence measurements to provide an estimate of the proportion of apoptotic cells within the population (apoptotic index).
The Incucyte® Nuclight Reagents for live-cell labeling have been validated for use with the Incucyte® Live-Cell Analysis System. They can be combined with the Incucyte® Caspase 3/7, Incucyte® Annexin V or Incucyte® Cytotox Dyes for multiplexed measurements of apoptosis and cytotoxicity in the same well.
Product
Qty.
Catalog. No.
20 μL
4440
20 uL
4704
4776
1 vial
4642
Incucyte® Annexin V Red Dye
4641
Incucyte® Annexin V Orange Dye
4759
Incucyte® Annexin V NIR Dye
4768
Incucyte® Cytotox Red Dye
5 μL x 5
4632
Incucyte® Cytotox Green Dye
4633
Incucyte® Cytotox NIR Dye
100 μL
4846
Incucyte® Nuclight Green Lentivirus (EF-1α, Bleo)
0.2 µL
4626
0.6 µL
4477
Incucyte® Nuclight Red Lentivirus (EF-1α, Bleo)
4627
4478
Incucyte® Nuclight Green Lentivirus (EF-1α, puro)
4624
4475
Incucyte® Nuclight Red Lentivirus (EF-1α, puro)
4625
4476
Incucyte® Nuclight Orange Lentivirus (puro)
4771
Incucyte® Nuclight NIR Lentivirus (puro)
4805
Incucyte® Cell Cycle Green/Orange Lentivirus (puro)
4809
Incucyte® Cell Cycle Green/Red Lentivirus (puro)
4779
Incucyte® Nuclight Rapid Red Dye
50 µL
4717
Incucyte® Nuclight Rapid NIR Dye
4804
Incucyte® Cell-by-Cell Analysis Software Module
1 module
9600-0031
Incucyte® Advanced Label-Free Classification Analysis Software Module
BA-04867
Incucyte® Nuclight Lentivirus Reagents enable efficient, non-perturbing, nuclear labeling of living mammalian cells.
Developments in advanced software tools for label-free analysis, including our first software module to use a neural network
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