Q: What factors should I consider when planning my antigen specificity experiment?
A: Our compatible partner Immudex offers oligo-barcoded dextramer reagents (the dCODE™ Dextramer®) for determining antigen specificity. This product consists of a dextran backbone with a series of antigen-presenting MHC molecules (for interacting with T-cell receptors), a fluorescent molecule (Phycoerythrin), and a DNA barcode that is compatible with our Immune Profiling Solution (see below):
This allows for the detection of antigen-specific T-cells through use with our Feature Barcoding technology. The general workflow for using dextramer reagents will consist of obtaining a single-cell suspension, labeling the suspension with the dextramers and antibodies of interest, washing the suspension, then flow-sorting for dextramer specific T-cells (optional) before proceeding immediately to the correct Immune Profiling Solution User Guide with Feature Barcoding.
Below are some factors that should be considered when planning an experiment with dextramers:
- What percentage of the cell suspension is interacting with my antigen(s) of interest? The percentage of T-cells in a diverse cell population that interacts with a specific antigen is likely to be very low (frequently less than 1%). Therefore, when looking for antigen-specific interactions, it is recommended to sort for both dextramer positive interactions and also T-cell markers in order to increase the signal and reduce background. Please see this figure from our application note 'Redefining Cellular Phenotyping: Comprehensive Characterization and Resolution of the Antigen-Specific T Cell Response' as an example:
In the above figure, you can see that it is difficult to detect specific antigen-TCR interactions in the unsorted samples; when sorting the cells for T-cell markers and dextramer-positive interactions, we see a much stronger signal.
- Do I plan to perform antibody labeling in addition to dextramer labeling (either for flow sorting or for use with Feature Barcoding technology)? It is entirely feasible to stain cells with both dextramers and antibodies, but a major workflow consideration to take into account is that dextramer staining needs to be performed first. Dextramers do not bind as strongly as antibodies, and thus staining with antibodies first or at the same time can lead to crowding out of the dextramer interaction with the TCR. Please see our Demonstrated Protocol on Cell Labeling with Dextramer Reagents for more information.
- Experience level with flow sorting: Flow sorting is an advanced sample preparation method. Therefore, we recommend that customers interested in performing this technique either have previous experience with flow sorting or have access to resources (such as a sorting facility) who can help with this procedure in order to increase the chances of success.
- Our Demonstrated Protocol on Cell Labeling with Dextramer Reagents provides an example gating strategy for flow sorting. Please keep in mind that these are recommendations and may not be applicable for all sample types/experiments.
- Compatibility of the MHC allele to target sample: Dextramer MHC allele and donor allele should match
- Controls: Some level of background staining is common when working with dextramers, so having a negative control for comparison purposes is very important. Immudex support can offer more guidance on the appropriate controls to select for your antigen of interest.
For additional questions on dextramers, we recommend reaching out to firstname.lastname@example.org for more information.