Dextramer® Controls – Best Practices in Research Call for the Best Controls in Experiments
Controls eliminate confounding factors, distinguish signal from background, and help troubleshoot protocols
Controls Are the Core of Every Experiment
Positive and negative controls are often an afterthought in the design of experiments. And yet, without them, results leave room for ambiguity. Only properly controlled experiments can concretely discriminate hypotheses.
Controls also help resolve technical issues in experiments:
Dextramer® Negative Controls help maximize signal-to-noise ratios and enable accurate monitoring of your positive cell population.
MHC I Dextramer® Negative Control staining allows to distinguish a MART-1-specific CD8+ T-cell population from background staining using flow cytometry.
Why Use Negative Controls?
MHC Dextramer® are powerful flow cytometry reagents that bind with high avidity to antigen-specific T cells. This is a result of multiple simultaneous interactions at the cell surface between peptide MHC monomers on the Dextramer® and TCRs that recognize them. However, weaker general protein-protein- and other interactions not directly related to antigen recognition result in background staining of cells with lower intensity.
Negative control Dextramer® reagents are commonly used to set the threshold between background and antigen specific staining to optimize the gating/sorting of positive cells by flow cytometry.
Negative controls are also important in single cell experiments involving barcoded dCODE Dextramer® reagents to define thresholds for identification of antigen-specific cells in downstream data analysis.
The Importance of Allele-Specific Negative Controls
Background staining can be allele-specific, in some cases related to the strength of the peptide-independent interaction between the MHC on the Dextramer® and CD8 on the T cell surface. In addition, background staining can also be donor-dependent.
We therefore advise the use of allele-matched antigen presenting MHC Dextramer® and negative control Dextramer®. We also recommend evaluating the background in every donor sample included in an experiment.
MHC Dextramer® Peptide Pool Negative Control
We have developed a new class of allele-specific negative control reagents based on a novel innovative design. We use peptide pools of enormous diversity to create MHC Dextramer® that differ fundamentally from our normal reagents.
Instead of presenting a single pMHC monomer, MHC Dextramer® Peptide Pool Negative Control are decorated with MHC monomers that all present different peptides. In addition, no two MHC Dextramer® Peptide Pool Negative Control molecules are likely to be composed of the same combination of pMHCs.
As a result, MHC Dextramer® Peptide Pool Negative Control are unable to bind to T cells by antigen-specific TCR engagement. Thus, they are ideal for the delineation of background-stained cell populations
MHC Dextramer® Peptide Pool Negative Control Performance
MHC Dextramer® Peptide Pool Negative Control were used to stain multiple allele matched donor samples, which were then analyzed by flow cytometry. Representative flow charts show the very limited background staining observed with these reagents.
In addition, allele-specific differences in background staining are appreciated by comparing the beak shaped populations of CD8+ T cells weakly stained with either HLA-A*01:01 or HLA-A*03:01 MHC Dextramer® Peptide Pool Negative Control (upper left quadrant), with the more homogenously stained cell populations achieved with HLA-A*02:01 and HLA-B*07:02 MHC Dextramer® Peptide Pool Negative Control reagents.
Order Dextramer® Controls
Couple your Dextramer® reagents with the right positive and negative controls. Immudex offers a range of Dextramer® Positive and Negative Controls including the new MHC Dextramer® Peptide Pool Negative Control with fluorophore labelling for your flow cytometry and immunohistochemistry experiments.
You can use the webshop below to order online. Alternatively, send an email to [email protected] specifying:
Catalog number or desired MHC allele-peptide combination
Fluorophore (BV421, FITC, PE, APC, or none)
One test is 10 µl reagent and sufficient to stain 1–3 x 106 lymphoid cells or 2–5 x 104 clonal antigen-specific T cells.
To order DNA barcoded dCODE Dextramer® controls for single cell analyses, please send an e-mail to [email protected] specifying:
Your desired MHC allele-peptide combination. See our list of available products below
Test size (25, 50 or 150 tests)
Please note: dCODE Dextramer® is available as a standard product with the PE fluorochrome, or with FITC or APC fluorochromes (or no fluorochrome) via Custom Solutions and Services.
Depending on your experimental setup, you can select between Dextramer® Negative Controls with MHC monomers presenting:
a peptide pool (allele-specific MHC Dextramer® Peptide Pool Negative Control)
an empirically derived peptide sequence (MHC I) known to give very low levels of background staining
a peptide sequence unrelated to your experiment
a CLIP peptide (MHC II)
Depending on your experimental setup, for a positive control, you can order MHC Dextramer® reagents that recognize epitopes derived from widespread human viruses such as CMV, EBV or Influenza. For maximal coverage, several reagents can be ordered and pooled together. For instance, our internal experiments showed that in the context of HLA-A*0201 healthy donors, staining with the three MHC Dextramer® specificities below, showed a coverage of ~95% or (15 out of 16 donors were positive for one or more of these specificities).
Our webshop does not include all products. Contact us or email [email protected] and we will search our larger internal database of products for you. We also provide custom alleles and specificities.
Don’t find a MHC Dextramer® Peptide Pool Negative Control for your desired allele? Please contact us and we will make it!
For custom MHC-peptide combinations, a one-time entry fee will be required to evaluate each unique peptide's ability to bind to the requested MHC allele using standardized peptide binding assays. If the refolding process is unsuccessful, only the entry fee will be charged as we will not be able to deliver a product to you.
Dextramer® Custom Controls
Your experimental setup may require an MHC allele that is not listed in our catalog or different specificities. Our Custom Solutions and Services Team can ensure you have the right controls for your research. Just contact us.
BV421 is equivalent to Brilliant Violet™ 421, which is a trademark or registered trademark of Becton, Dickinson and Company or its affiliates, and is used under license. Powered by BD Innovation.
