Releasable antibodies for label-free cells after cell sorting

The benefits

• Epitope of a marker becomes available again for downstream applications that require free epitopes (e.g. cell culture experiments and cell analysis)
• Fluorescence channel becomes available again for relabeling (e.g. microscopy applications)
• Sequential flow sorting cycles for the isolation of specific cell subsets

The basic principle

REAlease Releasable Antibodies allow for the removal of any labels from cells after cell sorting in just one simple step.

The technology relies on recombinantly engineered antibody fragments that are, unlike conventional antibodies, characterized by low epitope binding affinities.

A novel covalent conjugation approach allows for multimerization and fluorescent labeling of these antibody fragments. They form the so called REAlease Fluorochrome Complex which facilitates high-avidity cell binding that is comparable to conventional fluorochrome conjugates.

This novel and innovative conjugation chemistry allows for fast removal of the REAlease Fluorochrome Complex from the cell surface by adding the REAlease Release Reagent after cell sorting. This leads to disruption of the complex and thereby results in monomerization of the antibody fragments.

Due to their engineered low binding affinity, they spontaneously dissociate from the cell surface.

As a result, label-free cells can be obtained after cell sorting providing maximal flexibility for a wide range of applications.

Uncompromised fluorescent antibody staining and efficient label release from target cells

The staining performance of REAlease Antibodies is equivalent to conventional antibodies with comparable brightness and low non-specific binding.

The removal of REAlease Antibodies is highly efficient and selective with no detectable fluorescence signal remaining after the release step.

Additionally, the labeling efficiency in the relabeling step is equivalent to the initial staining demonstrating that epitopes become completely available again.

For release and relabel with REAlease Antibodies refer to the REAlease Support Kit.

Multiparameter immunofluorescent labeling and release 

The new covalent conjugation chemistry of the fluorochrome conjugates allows a reversible multi-parameter cell labeling.

To demonstrate the versatility of this technology, we developed REAlease Fluorochrome Conjugates for several cell markers that can be used for multiparameter panel stainings of e.g. pan T cells.

In this example, five-parameter cell labeling of peripheral blood mononuclear cells (PBMCs) with REAlease Fluorochrome Conjugates allows for clear definition and discrimination of T cell subsets.

After the addition of the REAlease Release Reagent all fluorochrome conjugates can efficiently be released.