Embark on a new era of cell-based research! For the first time, RT-IC empowers researchers to conduct true real-time binding kinetics directly on live cells, by seamlessly integrating biosensor technology with flow cytometry. RT-IC allows for real-time monitoring of binding events on the surface of live, individual cells, providing unprecedented insights and possibilities for studying cell surface receptors, membrane proteins, and complex cellular processes, in their native environment.
At 2bind, our team of experienced scientists delivers the full potential of heliXcyto® and RT-IC to provide comprehensive solutions for your cell-based research needs. We combine cutting-edge instrumentation with expert data analysis and interpretation to deliver accurate, reliable, and actionable results.
Real-Time Interaction Cytometry (RT-IC) presents a paradigm shift in cell-based research, enabling researchers to measure real-time binding kinetics and affinities on the surface of individual cells.
RT-IC utilizes biocompatible microfluidic channels to capture and immobilize single cells on the surface of the sensor chip. This unique approach allows for precise control of the cellular microenvironment while maintaining cell viability and functionality. By combining this cell handling capability with the proven biosensor technology of the helix® platform, RT-IC provides a seamless workflow for real-time analysis of biomolecular interactions in the context of living cells.
The RT-IC technology offers several key advantages over traditional cell-based assays:
Real-time monitoring:
Capture the dynamics of binding events as they occur on the cell surface, providing valuable kinetic information that is lost in endpoint assays.
Native cell surface:
Eliminate the need for potentially disruptive chemical modifications or immobilization technologies, preserving the native state of the cell and its receptors.
Single-cell resolution:
Analyze binding events on individual cells, revealing heterogeneity within cell populations and uncovering rare subpopulations.
High sensitivity: Detect even weak or transient interactions, broadening the scope of your research and enabling the study of challenging targets.
Association rate constant. Provides information on how fast complexes form; can be used for KD determination.
Dissociation rate constant. Provides information on how fast complexes dissociate; can be used for KD determination.
Equilibrium dissociation constant. Can be obtained by kinetic or classical equilibrium binding analysis. Provides information about the strength but not the dynamics of an interaction.
Functional affinity, representing the overall strength of an interaction. Influenced by the binding affinity, binding valency, and the structural arrangement of target and ligand.
Dose-response data. Ligand concentration that gives half-maximal response or half-maximal activity.
Bivalent binding interactions frequently result in biphasic kinetics and delivering two KD values. Relative amplitudes reveal which KD is more relevant.
The time required until the antibody amount bound to the cell surface is reduced to 50%.
Besides KD values, the cell surface antibody retention time should be considered for comparing different antibody candidates.
Analyze the concentration-dependent effect of an inhibitor on the interaction of the analyte with the cell surface.
Immunology: Investigate the interactions between immune cells and their targets, characterize antibody binding kinetics, and develop novel immunotherapies.
Oncology: Study the binding of cancer drugs to their cellular targets, identify novel therapeutic targets, and develop personalized cancer treatments.
Neurobiology: Explore the interactions of neurotransmitters and neuromodulators with their receptors, elucidate the mechanisms of synaptic transmission, and develop new treatments for neurological disorders.
RT-IC on the heliXcyto® platform revolutionizes cell-based analysis by seamlessly integrating flow cytometry with biosensor technology. Here’s a closer look at how it works:
Real-Time Interaction Cytometry (RT-IC) offers unparalleled advantages for studying biomolecular interactions in their most relevant context: the living cell.
Native Folding: Membrane proteins like GPCRs often exhibit complex folding patterns influenced by the cell surface. RT-IC preserves these native conformations, ensuring accurate measurement of binding kinetics unattainable with surface-based techniques.
Native Density and Mobility: Cell membranes are dynamic environments with varying receptor densities and lateral mobility. RT-IC directly captures these complex interactions, providing insights beyond the limitations of surface-based assays.
Native Co-Interactions: Many cell surface receptors interact with other molecules, including heterodimerization partners. RT-IC uniquely accounts for these co-interactions in real time, offering a comprehensive view of binding kinetics in the cellular context.
By studying biomolecular interactions directly on living cells, RT-IC provides a more physiologically relevant and comprehensive understanding of these processes, crucial for drug discovery, therapeutic development, and basic research.
Traditional flow cytometry offers valuable insights into cellular characteristics, but it falls short when it comes to real-time analysis of biomolecular interactions on the cell surface. Here’s why RT-IC is the superior choice for studying these dynamic processes:
Suspension and adherent cells: As in flow cytometry, suspension and adherent cells can be measured in RT-IC. Adherent cells have to be detached prior to the measurement.
Cells from 6 to 25 µm diameter: RT-IC chips are available with cages of different sizes to accommodate cells with diameters from 6 to 25 µm.
Tumor cell lines, recombinant cells, primary cells: RT-IC is possible with any cell expressing the target of interest. It can be tumor cell lines, transfected recombinant cells as well as primary cells, like T cells or NK cells.
Antibodies: Antibodies can be used as soluble analytes in RT-IC. Differences between binding kinetics measured with purified proteins and on cells can differ tremendously, reflecting changes in antigen conformation, avidity effects and/or the influence of third interaction partners on the cell surface.
Bi-specifics: Binding kinetics of bi-specifics targeting two cell surface receptors on the same cell can differ dependent on densities and ratios of the two target proteins, which are not necessarily evenly distributed on the cell surface. The effect can be measured in RT-IC.
Protein ligands: The measurement of any protein ligand to a cell surface receptor is possible in RT-IC. It might be of relevance for target validation or in a ligand-inhibition setup.
Small molecules: The signal is independent of the analyte’s size. Interactions of small molecules with cell surfaces can be measured as long as they are fluorescent and emit red or green light or can be labeled with a fluorophore.
The minimal number of target molecules on the cell surface depends on the binding kinetics, and in particular on the association rate of the analyte. Slow on-rates can be compensated by high concentrations of analyte. Analytes can also be labeled with more fluorophores, or detected with a secondary antibody, to increase the sensitivity. To reduced the analyte consumption, it is possible to incubate cells and analyte offline for several hours or over night, and to monitor and analyze exclusively the dissociation.
The constant domains of antibodies contain many lysines that are accessible for amine coupling without impacting the binding to the target protein, which typically involves exclusively residues in the variable domains. However, exposed lysine residues in HCDR3 might represent a risk factor, and binding of such antibodies after labeling should be confirmed in GCI or FACS.
Target-internalization is likely to influence the measured binding kinetics. To receive clean data, internalization can be chemically inhibited, or cells can be fixed by PFA prior to the measurement.
The detector can handle very fast on- and off-rates, and affinities from µM to pM range can be measured. Slow association rates in combination with low target expression levels might not lead to sufficient signal increase within the measurement time. In these rare cases, dissociation-only experiments should be considered, which allow the real time monitoring of analyte dissociation after an off-line incubation of analyte and cells.
Cells as targets: RT-IC uses cells as targets and omits the need of high quality purified recombinant proteins, offering solutions in cases where the target proteins cannot be produced and purified in sufficient amounts and/or native folding conformation. Binding interactions are measured on the cell surface including all potential obstacles the analyte might face in the native surrounding and enable a comparison of candidates under conditions that might be more relevant for performance in vivo.
Kinetic data: RT-IC delivers kinetic data in contrast to titration experiments in flow cytometry. Association and dissociation rates are important parameters to be considered for planning concentrations, incubation times and dosing schedules for in vitro experiments or in vivo studies.
Automation: The heliXcyto® platform is equipped with an autosampler, reducing the hands-on time and minimizing manual operations.
Cell imaging: A built-in reflected light microscope and a CCD camera enable a strict observation and logging of cell appearance to improve the interpretability of collected measurement data.
Dual-color detection: Parallel detection of green and red fluorescence enables multiplexing experiments and reveals cooperative and competitive effects of two analytes labeled with different colors.
