heliXcyto
Experimental Considerations
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.
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.
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.