10x Genomics does not currently support dCODE™ Dextramer®-only analysis. We recommend staining with dCODE™ Dextramer® reagents in conjunction with a small panel of TotalSeq C antibodies for antigen-specific T-cell analysis. This increases the cell calling accuracy of the data. Please contact Tech Support for TotalSeq C antibody recommendations.
Question: What factors should I consider when planning my antigen specificity experiment using dCODE™ Dextramer® from Immudex?
- What percentage of the cell suspension is interacting with my MHC-peptide of interest? The percentage of T-cells in a diverse cell population that interacts with a specific antigen is generally very low (frequently less than 1%). Therefore, when looking for antigen-specific interactions, it is recommended to enrich both dCODE™ Dextramer® positive interactions and T-cell markers to increase the frequency of antigen-specific cells in the cell sample. 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 dCODE™ Dextramer®-positive interactions, we see a much stronger signal.
- Do I plan to perform antibody labeling in addition to dCODE™ Dextramer® labeling (either for flow sorting or for use with Feature Barcoding technology)? It is entirely feasible to stain cells with both dCODE™ Dextramer® reagents and antibodies, but dCODE™ Dextramer® staining should be performed prior to antibody staining. Antibodies bound to their cell surface markers can represent a steric hindrance to the binding of dCODE™ Dextramer®. Please see our Demonstrated Protocol on Cell Labeling with dCODE Dextramer® for more information, or contact Immudex to learn more about the use of dCODE™ Dextramer®.
- 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 dCODE Dextramer® 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: The MHC allele displayed on the dCODE™ Dextramer® should match the donor’s MHC.
- Controls: It is recommended to include more than one negative control in the dCODE™ Dextramer® pool. Negative controls are used to evaluate the background threshold during data analysis. For more information about the choice of negative controls, contact firstname.lastname@example.org
For additional questions on dCODE™ Dextramer® technology, we recommend reaching out to email@example.com for more information.
Products: Single Cell Immune Profiling