Tipping point

We describe how to determine the so-called tipping point for a certain cell type - antibody combination with the CellVysion. 

Measuring of cells on a gradient ligand

The tipping point is the characteristic ligand density that is just enough to capture the cells/particles under certain shear conditions. This value of avidity determined as functional Rmax value depends both on the number of receptors and the monovalent affinity per cell. Therefore, this setup and method will allow for cell-antibody avidity determination. Once we determined the tipping point, we measured the ligand density at the tipping point.

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Cell avidity detection

The fluidics: The Cuvette Injection Flow (CIF), of the SPR imager of Vysens enables the injection of samples directly from a cuvette. This cuvette is placed on top of a two-channel flow cell. The analyte can be injected straight into the flow chamber, there are no air bubbles used for separating the running buffer from the sample. The CIF can be applied to generate a ligand density gradient in both flow channels. 

Large particles (e.g., cells) can be aspirated without clogging the fluidics. The SPR imaging system makes the sedimentation of the cells visible in real-time. In order to keep the sample volume low (e.g., 70μL), back-and-forth flow is applied.  Association times can be e.g., half an hour. 

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Gradients

The gradient makes all ligand densities available, from very high to low, up to zero. The zero-ligand density location is used as a reference signal to compensate for common mode signals such as bulk refractive index shifts, temperature effects, nonspecific binding to the hydrogel, etc. [10].

The CellVysion makes it possible to obtain the single molecular affinity parameters [11] from hundreds of biomolecular interaction events on the gradient. In the SPR imager, the generation of the gradient can be measured for both channels [12].

Cyto Vision 2

More unique applications possible in CellVysion

But there are more unique applications that have been realized in the CellVysion:

  • Cells are characterized for cell surface receptor avidity or strength of binding related to monophasic affinity in combination with the number of cell surface receptors. As stated before, in our opinion many scientists could benefit from the experimental advantages. We would be pleased to contact scientists who are interested in cooperation with us.

You just need a little to see it all.