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ACEA xCELLigence DP Bundle


Cell invasion and migration measured in real-time.

The xCELLigence® RTCA DP instrument uses noninvasive electrical impedance monitoring to quantify cell proliferation, morphology change, and attachment quality in a label-free, real-time manner. What distinguishes the DP (dual purpose) model from our other xCELLigence instruments is its ability to additionally make kinetic measurements of cell invasion and migration (CIM) using an electronically integrated Boyden chamber (CIM-Plate® 16). The three cradles of the DP instrument enable three separate electronic 16-well plates to be controlled and monitored in parallel or independently of one another, allowing maximal productivity for multiple users. The instrument is placed in a standard CO2 cell culture incubator and is powered and controlled via a cable connected to the control unit (a laptop computer) housed outside the incubator. User friendly real-time cell analysis (RTCA) software allows for real-time interfacing with all three cradles, and includes real-time data display and analysis functions.

By skipping the guesswork associated with end-point assays and eliminating the time- and labor-intensive steps of traditional methods, real-time cell analysis with the xCELLigence® RTCA DP vastly improves efficiency and overall productivity.



Positioned between reductionistic biochemical assays and whole organism in vivo experimentation, cell-based assays serve as an indispensable tool for basic and applied biological research. However, the utility of many cell-based assays is diminished by: (1) the need to use labels, (2) incompatibility with continuous monitoring (i.e. only end point data is produced), (3) incompatibility with orthogonal assays, and (4) the inability to provide an objective/quantitative readout. Each of these shortcomings is, however, overcome by the non-invasive, label-free, and real-time cellular impedance assay.

The functional unit of a cellular impedance assay is a set of gold microelectrodes fused to the bottom surface of a microtiter plate well (Figure 1). When submersed in an electrically conductive solution (such as buffer or standard tissue culture medium), the application of an electric potential across these electrodes causes electrons to exit the negative terminal, pass through bulk solution, and then deposit onto the positive terminal to complete the circuit. Because this phenomenon is dependent upon the electrodes interacting with bulk solution, the presence of adherent cells at the electrode-solution interface impedes electron flow. The magnitude of this impedance is dependent on the number of cells, the size and shape of the cells, and the cell-substrate attachment quality. Importantly, neither the gold microelectrode surfaces nor the applied electric potential (22 mV) have an effect on cell health or behavior.

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Figure 1.  Overview of cellular impedance apparatus.  A side view of a single well is shown before and after cells have been added.  Neither the electrodes nor the cells are drawn to scale (they have been enlarged for clarity).  In the absence of cells electric current flows freely through culture medium, completing the circuit between the electrodes.  As cells adhere to and proliferate on the electrodes current flow is impeded, providing an extremely sensitive readout of cell number, cell size/morphology, and cell-substrate attachment quality.


The gold microelectrode biosensors in each well of ACEA’s electronic microtiter plates (E-Plates®) cover 70-80% of the surface area (depending if a view area is present). Rather than the simplified electrode pair depicted in Figure 1, the electrodes in each well of an E-Plate are linked into “strands” that form an interdigitating array (Figure 2). This arrangement enables populations of cells to be monitored simultaneously and thereby provides exquisite sensitivity to: the number of cells attached to the plate, the size/morphology of the cells, and the cell-substrate attachment quality.

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Figure 2.  Impedance electrodes on ACEA’s E-Plates.  (A) Simplified schematic of the interdigitated electrodes used in each well of an E-Plate.  Electrodes are not drawn to scale (only a few are shown, and they have been enlarged for clarity).  Though cells can also be visualized on the gold electrode surfaces, the electrode-free region in the middle of the well facilitates microscopic imaging (brightfield, fluorescence, etc.).  (B) Photograph of a single well in a 96-well E-Plate.  (C) Zoomed in brightfield image of shadowed electrodes and unstained human cells.  (D) Gold electrodes and crystal violet stained human cells, as viewed in a compound microscope.


While it can run the same assays as the other xCELLigence instruments, the xCELLigence RTCA DP (dual purpose) model has the additional capability of performing real-time cell invasion/migration assays using ACEA’s CIM-Plate, which is essentially an electronically-integrated Boyden chamber.  As shown in Figure 3A, cells are placed in the upper chamber either directly on top of the microporous membrane (migration assay) or on top of a basement membrane matrix and/or cell monolayer previously deposited on the membrane (invasion assay).  Moving towards chemoattractant in the lower chamber, cells pass through the microporous membrane and then deposit onto gold impedance electrodes (which were described in the previous two sections of this technology overview).  This provides a very sensitive and reproducible continuous kinetic record of cell migration/invasion (Figure 3B).  Parallel assays using microscopic imaging to quantify migrated cells demonstrate a perfect correlation between the impedance signal of the CIM-Plate and the number of cells that have migrated.  Click the following links for Cell Migration/Invasion Application Overviews or Cell Migration/Invasion Publications.

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Figure 3.  Quantitative real-time analysis of cell invasion/migration.  (A) CIM-Plate detail.  The upper panel shows a cut away view of eight wells of the CIM-Plate.  The expanded view in the lower panel illustrates the upper and lower chambers for a single well.  The bottom surface of the upper chamber is composed of a microporous membrane that cells can migrate through.  Gold electrodes on the underside of this membrane detect the presence of adherent cells.  For a simple migration assay (not illustrated here) the cells being monitored would be plated directly onto the membrane.  For an invasion assay (shown here), cells are plated on top of a basement membrane matrix, a cellular monolayer, or some combination thereof.  (B) Real-time analysis of murine macrophage migration in the presence or absence of chemoattractant.  In the absence of cells (negative control; blue line) the impedance signal is unchanged over the 75 minutes of the assay.  Though some cells do migrate through the porous membrane in the absence of chemoattractant (green line), macrophage migration is significantly stimulated when chemoattractant is present in the lower chamber of the CIM-Plate (red line).  Figure “B” adapted from PLoS One. 2013 Mar; 8(3): e58744.


The impedance of electron flow caused by adherent cells is reported using a unitless parameter called Cell Index (CI), where CI = (impedance at time point n – impedance in the absence of cells)/nominal impedance value. Figure 3 provides a generic example of a real-time impedance trace throughout the course of setting up and running an apoptosis experiment. For the first few hours after cells have been added to a well there is a rapid increase in impedance. This is caused by cells falling out of suspension, depositing onto the electrodes, and forming focal adhesions. If the initial number of added cells is low and there is empty space on the well bottom cells will proliferate, causing a gradual yet steady increase in CI. When cells reach confluence the CI value plateaus, reflecting the fact that the electrode surface area that is accessible to bulk media is no longer changing. The addition of an apoptosis inducer at this point causes a decrease in CI back down to zero. This is the result of cells rounding and then detaching from the well bottom. While this generic example involves drug addition when cells are confluent, impedance-based assays are extremely flexible and can also evaluate the rate and extent of initial cell adhesion to the electrodes, or the rate and extent of cell proliferation.

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 Figure 3.  Generic real-time impedance trace for setting up and running an apoptosis assay.  Each phase of the impedance trace, and the cellular behavior it arises from, is explained in the text.

Moving beyond the generic example shown above, Figure 4 shows actual real-time impedance data acquired using E-Plates in ACEA’s xCELLigence® real-time cell analysis (RTCA) instruments. Figure 4A shows impedance traces for the first two hours after A549 cells have been added to an E-Plate, the wells of which were previously coated with differing concentrations of collagen IV. While Figure 4B demonstrates the change in cell index that occurs within the first few minutes of exposing HeLa cells to the GPCR agonist dopamine, Figure 4C evaluates NK cell-mediated cytolysis of cancer cells over the course of 20 hours. Figure 4D highlights the variety of changes that can occur in cell index depending on a drug’s mechanism of action.

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Figure 4.  Examples of real-time impedance traces obtained using E-Plates and xCELLigence RTCA instruments.  (A) Real-time monitoring of A549 cell adhesion to E-Plate wells that had been pre-coated with different concentrations of collagen IV.  Note the correlation between impedance values (Cell Index) and the number of adherent cells visible in the microscope.  (B) Real-time impedance traces for HeLa cells exposed to different concentrations of the GPCR agonist dopamine.  The black arrow indicates the time of dopamine addition.  (C) Real-time impedance traces for NK 92 cell-mediated cytolysis of MCF7 breast cancer cells.  (D) Real-time impedance traces for A549 cells exposed to drugs displaying a variety of mechanisms of action.


RTCA provides a quantitative readout of cell number, proliferation rate, cell size/shape, and cell-substrate attachment quality. Because these physical properties are the product of thousands of different genes/proteins, RTCA can provide an extremely wide field of view on cell health and behavior. Everything from endothelial barrier function and chemotaxis to filopodia dynamics and immune cell-mediated cytolysis have successfully been analyzed on xCELLigence instruments. Despite the breadth of their reach, xCELLigence assays are still capable of interrogating very specific biochemical and cellular phenomena. Appropriate use of controls and/or orthogonal techniques make it possible to correlate the features of an impedance trace with specific cellular/molecular phenomena.

The xCELLigence® RTCA DP system is one of the most versatile xCELLigence systems.  It is capable of performing all xCELLigence applications, except those which are cardio specific (analyzing cardiomyocyte contractile and electrical activities).  The RTCA DP system is the only xCELLigence system that supports chemotactic migration/invasion assays using CIM-Plate.  View the table to the left for additional applications that the RTCA DP system is compatible with.

Workflow of the xCELLigence RTCA DP System:  Cell Migration & Invasion
No cell labeling required, fully automated, physiological conditions

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Pre-defined protocols guide you through experimental set-up and analysis in seconds.

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Figure 1. Zoomed in screen shot of table for recording the contents/conditions of each well in an E-Plate.

For the xCELLigence RTCA DP, SP, and MP instruments experiments are programed and executed, and data is analyzed, using the RTCA 2.0 Software.  This software enables facile experiment setup and execution along with powerful data analysis, while still remaining efficient and intuitive.  A general synopsis of how the software is used to run and analyze an experiment is shown below.

Step 1: Record Plate Layout
Using an intuitive graphical interface the contents/conditions of each well in the electronic microtiter plate (E-Plate®) are recorded (Figure 1).  Information fields for the wells include parameters such as cell type, cell number, drug identity, drug concentration, etc.  Table autofilling functions, similar to what are available in Excel or other spreadsheet programs, enable rapid data entry and automatic establishment of drug concentration gradients, cell number titrations, etc.  Even when multiple cell types and assay conditions are being examined, it takes just minutes to record the information for all the wells of a plate.

Step 2: Define Data Acquisition Parameters
Using a second table the details of data acquisition are defined.  These include the frequency of impedance recording and the experiment duration.

Step 3: Running the Experiment
Press “Run” and watch as impedance data is acquired in real-time for every well in the plate.  Even as data is being acquired it can be viewed, graphically manipulated, analyzed, and exported.

Step 4: Data Plotting and Analysis
Using an intuitive graphical interface the real-time impedance data for all the wells, or a subset of wells, from the E-Plate can be plotted (Figure 2A).  Data from multiple wells can be averaged and the coefficient of variation automatically calculated and plotted.  The viewing window for the x- and y-axes can be readily adjusted, and data traces can be normalized to a specific time point (immediately before drug addition, for example).  Lastly, curve fitting functionalities enable calculation of rates of change, EC50 values, etc. (Figure 2B).


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Figure 2.  Data plotting and analysis using the RTCA 2.0 Software.  (A) Screen shot of data plotting/analysis window.  Here all of the curves have been normalized to the time point immediately preceding drug treatment (denoted by the bold back vertical line).  Error bars represent coefficients of variation.  (B) Dose-response curve.  Plotting cell index values (at a specific time post drug treatment) as a function of drug concentration enables determination of an EC50 value.  These types of calculations are readily performed using built in data analysis functions.