Real-Time Interaction Cytometry (RT-IC)
Real-Time Interaction Cytometry (RT-IC) is a novel technology applying principles of biophysics and cytometry to enable the measurement of real-time kinetics directly on cells. The unique heliXcyto chip allows for the instantaneous and reversible immobilization of any eukaryotic cell type on the chip surface based on mechanical retention. After the trapping of single cells, association and dissociation of fluorescently labelled analytes can be monitored in real-time due to highly sensitive fluorescence detection with single-photon counters.
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The following parameters can be measured from analytes binding directly to target cells:
Association rate kon
Dissociation rate koff
Affinity, avidity, stability, Kd, t1/2
Fluorescence signal amplitude Fcps, relative target density
You can analyze any analyte of your choice with RT-IC, from small molecules to cytokines to bispecific antibodies to antigen-MHC complexes. The only prerequisite is that it needs to be fluorescently labelled.
No, RT-IC measurements can only be performed with a heliXcyto biosensor. The heliX+ is optimized for the measurement of stably immobilized molecules on the biochip. The heliXcyto has to fulfil the requirements to measure living cells and therefore, specific cytometry-related adjustments needed to be integrated into the biosensor concerning fluidics and configuration. These changes make sure that you will get the accuracy and precision you need to measure kinetics on cells while at the same time providing the necessary conditions to protect precious cell samples. Contact us for attractive package deals for both instruments.
Yes, you can differentiate affinity and avidity and even quantify the contribution of different binding modes, for example binary and ternary binding modes of antibodies.
Instrument and Chip Handling
The cell traps are made from a bio-compatible polymer. If polymer-interaction is of concern, they can be passivated with a layer of BSA protein.
heliXcyto chips can be re-used many times. An automatic regeneration step is washing out your trapped cells after each measurement. Additional chip cleaning by an optional Chip wash is recommended. Extensive cleaning is also possible manually on the bench with our Chip cleaning kit. You only need to switch to a new heliXcyto chip if your background fluorescence exceeds the cut-off of 3 x 105 cps in the Cyto chip test and cannot be lowered by a manual chip cleaning anymore, or if the traps got lost.
Different maintenance scripts for the heliXcyto are provided in heliOS. Optimally, you include a Cyto chip wash after each measurement and 1-3 System washes at the end of the entire experiment. Regular Clean & Sleep routines of the heliXcyto keep the device clean.
To assure chip longevity, handle it with care and avoid extreme temperature jumps. Do not freeze the heliXcyto chip. After use or if you observe increasing fluorescence background counts (above 3 x 105 cps), clean the chip with the Chip cleaning kit and store it at 4 °C in the fridge. You can find further handling tips on the information card delivered together with the heliXcyto chips.
The normalization peak should be a sharp injection and ejection of Normalization solution, with a plateau ideally in the same fluorescent count range as the highest measurement data. If the normalization peak injection or ejection is not precise the tube passivation could be uneven, air bubbles might interrupt the fluorescent solution, a pump could have stopped working, or the normalization solution could be too old. First, prepare a fresh normalization solution in RB1. Make sure you have added detergent to the running buffer. We recommend 0.01 % Pluronic, which is suitable for living cells. If that does not solve the issue, restart the device.
New heliXcyto chips have a raw fluorescence below 3 x 105 cps in the measurement channel when performing a chip test (LED = 0.5). Stable adherence of fluorescently labeled analytes to the chip material (glass, gold, polymer) might lead to an increase of this background fluorescence which can be noted by a difference of raw fluorescence counts before and after the measurement, as well as increasing signals in repeated chip tests. As the single photon counters are damaged by too high fluorescence, the shutter might close for protective reasons if the analyte signal on top of a high chip background reaches more than 2 x 106 cps. In this case you can lower the LED power in your next run, or reduce the analyte concentration. Make sure to clean the chip before storage with the Chip cleaning kit to prevent excessive fluorophore build-up.
There are several ways to avoid a drift of the signal. First, lower the LED excitation power to below 0.5. If the drift is already visible in the baseline, you can perform a Cyto chip test which includes a bleaching step. If the signal still drifts, you should thoroughly clean the device by performing 1-3 x System Washes. Do not use the camera during a measurement.
My signal is remaining at a high level even during the dissociation phase (dissociation signal not returning back to baseline).
Consider the measurement time in relation to the koff rate. Maybe you are working with a very strong binder and need to extend the dissociation phase. A technical reason might be the capture of autofluorescent dirt particles (often visible in the snapshots). Make sure all bottles, vials and microtiter plates are free from dust (if necessary, rinse them with water before use) and filter or centrifuge all buffers and solutions before use. If a malfunction of the buffer pump is the reason (no washout of analyte, which is visible in the raw data on both spots), please try to restart the device.
Ensure that your cell suspension is monodisperse (no clumping) and has a concentration of 1 x 105 cells/mL or higher. Resuspend the cell suspension freshly before inserting the samples into the heliXcyto biosensor. You can filter the cell suspension through a cell strainer to reduce clumping. Especially when working with small cells, increasing the cell concentration up to 10 x 105 cells/ml may enhance the capture efficiency.
Thoroughly wash the cell suspension before the measurement and resuspend it by pipetting up and down or pass it through a strainer/filter. In order to avoid aggregation, you can also dilute the cell suspension. Use buffer without Ca2+ and Mg2+ such as our RB1 and/or add EDTA.
The sample tray / chip tray is stuck or the software is not detecting its position correctly. What can I do?
Restart the device. If this does not help, contact our customer support. As a general note, you should always insert the sample tray and the chip tray completely, i.e. do not instantly stop once you encounter a slight resistance
The heliXcyto takes care of this for you. The only step you have to do manually is to harvest your cells, wash and count them, and place them in the autosampler of the heliXcyto. The cells are then taken up automatically and will get trapped in cell traps on the heliXcyto chip. You do not need to grow the cells directly on the biochip or modify them in any way. You do not need to screen your cells for specific surface molecules for capturing, since they will be physically retained in the cell traps.
The integrated CCD camera of the heliXcyto takes a snapshot of the sensor spot after the trapping, thus you can visually control the successful immobilization of cells.
You can use any cell type of your choice for RT-IC measurements. We provide heliXcyto chips with different trap sizes suitable for cells from about 6 to 25 µm diameter. There is no precise size limit for eukaryotic cells, since this is also highly dependent on the flexibility of your cell type. If you are not sure whether RT-IC is suitable for your cell type, you can simply give it a try.
Yes. You simply have to detach them before and put the cell suspension into the autosampler of the heliXcyto. We recommend buffer without Ca2+ and Mg2+ or addition of EDTA for the handling of adherent cells.
Yes, your RT-IC sample should be a single-cell suspension.
There is no need to fix the cells, you can perform RT-IC measurements directly after cell culture with living cells. However, you can also use fixed cells, if preferred.
The integrated light microscope and CCD camera allow for the visual control of your cells using snapshots. Viability stains in the red or green range can be used for survival optimization.
The integrated light microscope and CCD camera allow for the visual control of your cells using either snapshots or video recordings. Video recordings might be helpful if you expect morphology changes of your cells in flow or if previous data suggests any other time dependent morphological changes.
RT-IC can detect low-expression molecules down to a density of about 1000 molecules / cell. The exact detection limit depends on the analyte labeling and affinity.
Due to optimized fluidics you only need 35 μl of cell suspension per run with a cell concentration of about 1 x 106 cells per ml.
The dissociation script records only the dissociation of your analyte. In a dissociation experiment, the cells are pre-incubated with the labeled analyte before being transferred into the autosampler of the heliXcyto (comparable to immunostainings for FACS or microscopy). This method reduces measurement time and sample consumption and guarantees maximum binding signal. It is therefore ideally suited if you aim for higher throughput and want to screen many analytes for their binding capacity and stability. If your research question involves the association rate of your molecule, please choose the kinetic script.
Both the sample tray (where samples are stored before injection) as well as the chip (where the measurement takes place) are independently temperature-controlled. The chip temperature can be adjusted to 15 °C – 40 °C. The temperature range for the sample tray is 4 °C – 40 °C. Please take into account that the cell sample and the analytes are stored in the same sample tray before measurement when deciding on a storage temperature.
No, the heliXcyto does not include an incubator. We recommend using a well buffered solution for your cell suspension, that is not dependent on CO2 buffering. Furthermore, you can adjust the temperature of the sample tray to any temperature between 4 °C – 40 °C.
RT-IC measurements can be run in any media or buffer of your choice, just make sure to avoid pH indicator dyes such as phenol red. In order to ensure good cell viability throughout the assay it is important to keep them at a physiological pH at all times. Since the heliXcyto does not include an incubator, we recommend using a well buffered solution (e.g. PBS based) for your cell suspension that is not dependent on CO2 for maintaining a physiological pH.
Currently, this is not possible.
RT-IC can discern off-rates > 1 s-1.
Bleaching of the fluorophore is reduced by:
- a very low excitation LED power
- the usage of stable fluorophores
Dyes used in our labeling kits show hardly any signal drift of bulk fluorophore in the microfluidic channel over 1 hour continuous excitation. However, you might consider performing specific controls when measuring slow dissociations over many hours. Our application specialists are happy to discuss specific solutions with you. Contact us here.
Each heliXcyto module allows an automated throughput of about 15 full kinetic experiments, depending on chosen contact times. You can scale this up according to your specific high-throughput needs by combining several modules in one network. Contact us for heliXcyto package deals.
Labeling of your analyte can be done either by direct conjugation of a fluorescent dye or by secondary detection. We offer a labeling kit based on amine-reactive NHS-ester dyes. Alternatively, you can use any other labeling method of your choice, keeping the detection ranges of the heliXcyto in mind.
The heliXcyto has a green and a red detection channel. You can use any low-bleaching fluorochrome with an excitation wavelength in the range of 490-510 nm or 605-625 nm and an emission wavelength in the range of 525-575 and 655-685 nm, respectively.
The automated workflow of the heliXcyto includes snapshots of the electrodes before and after cell immobilization, after the dissociation as well as after trap regeneration. These can be evaluated during data analysis.
It is recommended to reproduce measurements generated on single-trap chips multiple times to get a good estimate of cell-to-cell-variability. Data obtained on multiple single cells can be plotted into one graph to get an overview of your cell population. Alternatively, a fast way to achieve population wide average kinetic rates is the measurement on chips trapping multiple cells in 5 traps on the electrode in each run.