--- title: Be positive! 5 ways to confirm your MHC tetramer is binding description: "Follow these 4 elements in choosing the best negative tetramer control: 1. Same fluorochrome 2.Same MHC allele 3. Peptide w/ no reactivity in your system 4." image: https://blog.mblintl.com/hs-fs/hub/424171/file-2569781963.png --- **Please Note:** MBL International will be shutting down its operations effective December 31, 2024. Distribution of MBL products in the United States will be transferred to Cosmo Bio US on January 1st while European Distributors will remain unchanged. For any US inquiries regarding orders or support during this transition, reach out to Cosmo Bio: [https://www.cosmobiousa.com/](https://www.cosmobiousa.com/). For Non-US inquiries reach out to MBL in Japan: [https://www.mblbio.com/](https://www.mblbio.com/). 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[Home](https://blog.mblintl.com/) - [Blogs](https://blog.mblintl.com) - Be positive! 5 ways to confirm your MHC tetramer is binding # Be positive! 5 ways to confirm your MHC tetramer is binding ### Topics - Select Topic - 3D Cell Culture Advancements in Metabolism Research Allergy Antibodies Antibody Antibody Reagents Antibody-Peptide Conjugates Apoptosis Assay and Kits Autoimmune Disease Autophagy BRIC Benefits of Tetramer Assays Biomarker Cancer Cell Biology Cell Culture Reagents Cell Stabilization Custom Peptide Synthesis Cytokine Dextramer Staining Drug Discovery ELISA Epigenetics Epitope Tag Exosome Exosomes FCM Fc blocker Flow Cytometry Flow Cytometry Antibodies Fluoppi Fluorescent Proteins Future of Tetramer Technology Gastric Inhibitory Peptide Gene Regulation Gene Therapy Green Fluorescent Protein IL-18 Immune Response Immunology Inflammation Isotype Control Kits and Assays LC3 Liquid Biopsy MHC MHC Alleles MHC Class II Tetramers MHC Peptides Contribute MHC Tetramer MHC Tetramer Protocol MHC tetramer advancements Metabolism Mitophagy NCP Neuroscience Neuroscience Reagents Organoid PCSK9 PCSK9 ELISA Kit Peptide Peptide Exchange Peptide Synthesis Methods Peptide Synthesis Service Peptide-Infused Skin Care Products Preservation Purify exosome Quality Control QuickSwitch Quickswitch™ Custom Tetramer Kits RBP RIP Chip RNA Recombinant Protein Production Recombinant Proteins Regulation RiboTrap S100 Proteins SARS-CoV-2 T-Cell Detection TCR Tetramers Tetramer Tetramer Analysis Tetramer Assay Tetramer Assay Protocol Tetramer Staining TransFix Vectors Western Reagents atg8 covid-19 gfp keima red kinases mitochondria cell death p62 pathways phosphorylation pro IL-18 reagents rfp t cell tuberculosis ultracentrifugation vaccine ### Top Posts ### Subscribe to our Blogs Published by [Cheryl A. Guyre, Ph.D.](https://blog.mblintl.com/author/cheryl-a-guyre-ph-d) on Apr 17, 2015 4:35:17 PM ![](https://blog.mblintl.com/hs-fs/hub/424171/file-2569781963.png) In the previous [Tetramer Tips blog](https://blog.mblintl.com/tetramer-tips-for-success-how-to-be-a-negative-control-freak), I suggested ways for you to be a negative Control Freak. Now it’s time to think positive! A **positive control** for a tetramer is a sample that contains cells expressing the specific T cell receptor of interest, i.e. has the exact specificity of the tetramer. Okay, brace yourselves; I’m going to be frank here.  Having a positive control for tetramer experiments is often a quest for the Holy Grail. In many (most, in fact) cases, you will not have access to a positive control, unless one of your experimental samples *happens* to show a positive result. In an experiment where no positive events are seen and no positive control was used, you cannot necessarily conclude that the donor/patient/mouse is negative for that T cell specificity, because, heck, maybe you got distracted while pipetting and forgot to add the tetramer to your staining cocktail!  A tetramer experiment with no positive control and no positives in the experimentals is, therefore, uninterpretable. This is the hard truth we must face. Before you slip into despair about this, there *are* ways to obtain or create positive controls: 1. Positive control cells for certain specificities are available.  A notable example is the OT-I mouse, referenced in the previous tetramer blog on negative controls. Splenocytes, lymph node cells, or blood from this mouse are suitable positive controls for C57BL/6 mice that have been treated or immunized some way to generate a T cell response specific for H-2Kb OVA (SIINFEKL), as the OT-I mouse is transgenic for the T cell receptor that binds specifically to H-2Kb OVA (SIINFEKL).[![TetramerCTA1](https://blog.mblintl.com/hs-fs/hub/424171/file-2569781963.png?width=276&name=file-2569781963.png)](https://info.mblintl.com/tetramer-staining-guide) 2. Positive control cells can be generated *in vitro*.  The Mixed Lymphocyte-Peptide Culture (MLPC) method uses peptide-stimulation and cytokine culture to induce a specific T cell response.  Protocols can be found on pp. 14-15 of the Tetramer Staining Guide. 3. Positive control cells can be generated *in vivo*.  Check the literature to determine conditions that lead to a tetramer-specific response in your mouse strain and/or use vaccination conditions from your own experiments that worked previously.  Yes, it’s a bit more work, but if you don’t include that control and you get no positive events in your experiments, what do you have?  You got it: Uninterpretable data. 4. In the course of your tetramer studies, you may find donors or patients reactive to your tetramer of interest.  If you find this (and it is ethical, safe, and legal to do so), bleed the heck out of them!  Is it your fellow grad student or lab mate?  Perhaps she is willing to donate a leukopheresis pack to be used by researchers for years to come. (I believe some vials of my own cells may be in the back of some LN2 freezer at my alma mater. My proud legacy!) 5. Some will suggest you use a tetramer of a different specificity that’s positive in a patient population.  For example, tetramers to influenza, CMV, or EVB have been recommended as positive controls (in patients known to be reactive) for, say, a tetramer study looking at Mart-1-specific T cells.  While this is not ideal, as it is not a *tetramer* positive control, it will serve as an *assay* positive control to help you understand if other parts of the assay worked (wash steps, staining buffers, etc.)  You still could not conclude for certain that a negative result means the patient lacked Mart-1-specific cells, unless in the same assay a different patient had a positive hit with the Mart-1-specific tetramer. We researchers are trained and driven to **control** and **predict**, which is particularly handy for rare events flow cytometry with tetramers. Just remember to use these skills for good, not evil: when you go home at the end of the day, be sure to leave the Control Freak behind in the lab, planning the next experiment.   Tags: [Tetramer](https://blog.mblintl.com/topic/tetramer), [Immunology](https://blog.mblintl.com/topic/immunology) --- [Back to Blog](https://blog.mblintl.com) --- ### Comments Or Suggestions ## Related Articles ### Sign up for our latest news and promotions. 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