xCELLigence RTCA S16
Real Time Cell Analyzer – 16 well

Continuously monitor live cell proliferation, morphology and viability with label free assays in a 16 well format.

Cellular Impedance Explained

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.

xCELLigence Tech Overview Fig 1

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.

Impedance Electrodes

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.

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.

Real-Time Impedance Traces Explained

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.

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.

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.

Correlating Impedance with Cellular Phenomena

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. To learn more about how this is done, and to witness the sensitivity and versatility of the xCELLigence RTCA technology, peruse the many specific applications that are highlighted here.



Download the RTCA Software Lite

Free Trial. Compatible with xCELLigence S16 and iCELLigence systems.

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

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

For the xCELLigence RTCA S16 instrument, experiments are programed and executed, and data is analyzed, using the RTCA Software Lite. 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).

A. B.

Figure 2. Data plotting and analysis using the RTCA Software Lite. (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.


DescriptionCat. #
xCELLigence RTCA S16 – Bundle (complete system)00380601310
xCELLigence RTCA S16 – Instrument00380601300
xCELLigence RTCA S16 – Control Unit (laptop with pre-installed software)00380601430


DescriptionCat. #
E-Plate 16 (6 plates)05469830001
E-Plate 16 (36 plates)05469813001
E-Plate VIEW 16 (6 plates)06324738001
E-Plate VIEW 16 (36 plates)06324746001
E-Plate VIEW 16 PET (6 plates)00300600890
E-Plate VIEW 16 PET (36 plates)00300600880
E-Plate Insert 16 (16 well insert housed in a Receiver Plate that includes a lid; 6 of these assemblies are included)

·         (Inserts can be cultured in the non-electronic Receiver Plate prior to being transferred to an E-Plate for real-time cell analysis.)


xCELLigence® Immunotherapy Kit – B Cell Killing Assay

DescriptionCat. #
Complete Kit (E-Plates, Tethering Kit, Immunotherapy Software)8100004
Tethering Kit + Software8100006
Tethering Kit8100005


Application Notes


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Applications Overview

The xCELLigence® RTCA S16 system is compatible with the 16-well microtitor E-Plate®. This format/throughput is popular for dose dependent studies. The S16 system is capable of performing all xCELLigence RTCA applications, except cell migration and invasion assays (using ACEA’s CIM-Plate®) and cardio specific assays (analyzing cardiomyocyte contractile and electrical activities).

Workflow of the xCELLigence RTCA S16 System
No cell labeling required, fully automated, physiological conditions