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Real-Time Proliferation Assays

Cell proliferation is crucial to a myriad of biological processes. While proliferation drives organismal growth and the repair of injury, its dysregulation is associated with disease states such as autoimmunity and cancer. Cell proliferation is also commonly used as a generic indicator of cell health to shed light on other phenomena. Examples include tracking the proliferation of host cells as a way to detect the presence of a virus or using proliferation as a readout for drug toxicity.

Typical proliferation assays require significant manual handling time and provide limited endpoint data.

Explore Agilent xCELLigence real-time cell analysis to learn about a more comprehensive real-time proliferation assay that provides:

  • Direct measurement of cell health and proliferation
  • Quantitative and reproducible data
  • A simple and high-throughput method
  • Detailed insight into cellular mechanism of action
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Multiple Perspectives into Cell Health to Increase Confidence

Typical proliferation assays require significant manual handling time and provide limited endpoint data. The xCELLigence RTCA eSight delivers deeper insights into cell health and viability. Tracking cell health simultaneously by impedance and live cell imaging provides a primary and confirmatory result within the same assay, without increasing the workload. Automatically quantify and visualize cell number, attachment, and morphology over the course of multiple days.


Multiplex Impedance-Based Data with Live Cell Imaging

The xCELLigence RTCA impedance technology measures the early stages of proliferation by sensitively detecting changes in cell attachment strength to the well. While proliferation results by impedance and % confluence by imaging are consistent over the course of 50 hours, impedance captures and rank orders the impact of varying serum concentrations within 10 hours. Accurately measuring critical quality attributes at earlier time points can be especially valuable during cell therapy manufacturing.


Tracking Proliferation by Actual Cell Counts Using Fluorescent Dye-Labeled Nuclei

Agilent eLive Red and eLive Green dyes can be used to fluorescently label nuclei to enable actual cell counts on the xCELLigence RTCA eSight. When included in the growth media, the dyes display extremely low background fluorescence. In the presence of cells, these dyes diffuse through the plasma membrane and bind to dsDNA, which increases their fluorescence >1,000 fold and enables distinct nuclei to be counted. Importantly, even when imaged at high temporal frequency over multiple days, these fluorescent dyes are nonperturbing.


Tracking Proliferation by Actual Cell Counts Using Fluorescent Protein-Labeled Nuclei

Stable fluorescent cell lines can be produced using Agilent eLenti Red, eLenti Green, and eLenti Blue reagents. Upon integration into the host cell’s genome, these lentiviruses employ an EF1α promoter to constitutively express nuclear-localized mKate, EGFP, or TagBFP, respectively. Example images, displayed with and without eSight fluorescent nuclei masking, are shown for three common cell lines.


Analyzing Proliferation of Mixed Populations of Cells

As an example, A549 cells expressing EGFP were mixed with HT-1080 cells expressing mKate at a 1:1 ratio before seeding in E-Plate wells. Through the simple application of red and green nuclear recognition masks, eSight clearly demonstrates the proliferative advantage that the HT-1080 cells have over the A549 cells within this mixed population. This same approach can readily be applied to track the proliferative properties of each cell type present in studies of the tumor microenvironment or immune cell-mediated killing.



Monitor cell proliferation in real time

Typical biomarker-based assays require multiple manual handling steps and only yield endpoint measurements. Read the application note to learn how to increase the information richness of your proliferation assay without increasing the workload.

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New Developments in Cell Health, Viability, and Function

In one instrument, capture sensitive changes in cell number, movement, morphology, and attachment by simultaneous impedance monitoring and live cell imaging. Download the xCELLigence RTCA eSight Live Cell Analysis Brochure to learn more.

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