Agilent xCELLigence RTCA HT - High Throughput

RTCA Analyzer

Overview

The xCELLigence RTCA HT instrument uses biosensors to continuously monitor cell proliferation, morphology change, and attachment quality with a label-free protocol in a 384-well format for high throughput screening experiments, such as antibody screening. Observe the real-time effects of therapeutic antibodies on live cells.

Integrate up to 4 instruments controlled by a single control unit for 1,536 wells to meet high throughput sample screening needs. The RTCA HT model can be used as a single instrument or integrated into a multi-instrument high-throughput workflow with automated liquid handling.

• Label-free real-time cell analysis for high throughput screening of therapeutic antibodies, drug compounds, and cytotoxicity.
• Obtain continuous cell assay data points during the entire experiment.
• RTCA provides a continuous quantitative readout of cell number, proliferation rate, cell size/shape, and cell-substrate attachment quality.




Video


Agilent xCELLigence RTCA HT - High Throughput



The xCELLigence High Throughput (HT) model offers a quick and simple way to continuously monitor cell proliferation, morphology change and attachment quality for high throughput screening experiments. The platform offers a 384-well format. For even higher throughput demands, up to four instruments can be integrated and controlled by a single control unit. can be integrated with up to four instruments to meet high throughput screening needs

Typical Applications
• Cancer immunotherapy:
With the monitoring of cell killing

• Virology & Infectious diseases:
Monitor pathogens behaviour

• Cell signalling:
Capture changes in cell behaviour due to signalling

• Cytotoxicity Overview:
Monitor cell behaviour and attachment to the plate surface

• Cell Barrier Function:
Continuous monitoring of the cell barrier

• Cell Adhesion:
Studying cell adhesion and cell spreading

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.



Functional Unit of 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


xcelligence
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 Left: 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 Right: 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.
xcelligence


xcelligence
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.

Figure Left: 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.
xcelligence





How is xCELLigence RTCA used for killing assays? The xCELLigence Real-Time Cell Analysis (RTCA) instrument monitors cell killing in real time. Simply add target and immune effector cells to the patented microtiter plates (E-Plates), load the instrument, and start reading. The biosensors embedded in the bottom of each well allow for the automated kinetic measurement of changes in cell number, size, and substrate attachment quality.

Target cell death can be measured continuously and automatically from hours to days without the use of labels.





Data Analysis & Real-Time Data Acquisition Using RTCA HT Software
RTCA HT Software is used for operating the RTCA HT Instrument. It provides capabilities for flexible experiment setups, real-time data acquisition, and powerful data analysis functions.

Run an experiment and analyze the data in the following 4 simple steps.
Step 1: Record Plate Layout
Step 2: Define Data Acquisition Parameters
Step 3: Running the Experiment
Step 4: Data Plotting and Analysis

The RTCA Software Pro Immunotherapy module is now compatible with RTCA HT data. For more detailed analysis for immune cell killing assays.
xcelligence
Cancer Immunotherapy
Immune cell-mediated tumor cell killing can involve the components of both the innate and adaptive immune systems, including natural killer (NK) cells, cytotoxic T cells (MHC-dependent), antibodies secreted by B lymphocytes, engineered antibodies such as bispecific antibodies and bispecific T cell engagers (BiTEs), and genetically engineered T cells targeting specific tumor antigens (e.g., CAR-T, MHC-independent). The xCELLigence Real-Time Cell Analysis (RTCA) instrument monitors cell killing in real time. Simply add target and immune effector cells to the patented microplates (E-Plates), load the instrument, and start reading.
xcelligence



Virology & Infectious Diseases
Study pathogen fitness and behavior in real-time by using xCELLigence Real-Time Cell Analysis (RTCA) instruments. Continuously monitor viral infections and bacterial biofilms in an automated, non-invasive, and label-free manner.
xcelligence



Cell Signaling
xCELLigence Real Time Cell Analysis captures changes in cell size, shape, and proliferation rate in response to membrane receptor signaling. Label-free and continuous monitoring provides insights in cell response in the initial minutes of exposure and their response over days.
xcelligence



Cytotoxicity Overview
xCELLigence real time cell analysis (RTCA) uses biosensors on the bottom of E-Plate wells to continuously monitior the number of cells present, their size/morphology, and how tightly they are interacting with the plate surface. Cytotoxic responses nearly always involve biochemical changes that directly, or indirectly, affect one or more of the these parameters. Consequently, xCELLigence is able to monitor cytotoxic responses resulting from an exceptionally wide range of molecular targets (Figure 1).
xcelligence



Cell Barrier Function
The impedance-based xCELLigence Real Time Cell Analysis (RTCA) assay provides a label-free automatic alternative to solute permeability assay and transendothelial electrical resistance (TEER) assay. RTCA assays provides continuous monitoring of cell barrier functions in response to disease state, inflammation, and compound treatment.
xcelligence



Cell Adhesion
xCELLigence Real-Time Cell Analysis (RTCA) instrument continuously monitor cell adhesion and spreading without any manipulation of cells. Study interactions between extracellular matrix (ECM) proteins and cell surface integrins in an automated, non-invasive, and label-free manner.
xcelligence
Analyzer Application Adhesion Apoptosis Cell Characterization
Cytotoxity Immune Cell Killing Proliferation
Receptor Signaling Stem Cells Virus Cytopathic Effects
Depth 13.5 cm
Height 13.5 cm
Operating Environment Relative Humidity 5-98 %
Operating Environment Temperature 15-32 °C
Sampling Format 384-well plate
Width 16.5 cm