The Lab Notebook

Gating strategy for tetramer analysis to eliminate non-specific event

Posted by Wushouer Ouerkaxi, PhD, MD on Nov 14, 2018 1:00:00 PM

While MHC tetramer staining of antigen specific T cells for flow cytometry is considered highly specific, it could still be associated with non-specific staining. The gating strategy shown below is an example that investigators have used to reduce non-specific staining events using a dump channel. A dump channel can be planned on a case by case basis, on the experimental design, and consists of a pool of antibodies against cells that do not express our specific targets. For example, CD8+ T cells, anti-CD4/anti-CD14/anti-CD19 may be used to eliminate these immune cells from our targeted gate, and all may be labeled with the same fluorochrome. 

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Tags: Tetramer, Flow Cytometry

Fc blockers to reduce background in Tetramer staining

Posted by Tomohiro Nakajo on Sep 25, 2018 1:00:00 PM

Fc blockers are needed for monocytes, macrophages, B cells and granulocytes. Since T cells normally do not express Fc receptors, some investigators do not use Fc blockers and instead use a dump channel (e.g. CD14/CD19) during flow cytometry analysis to exclude Fc-expressing populations. 

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Tags: Tetramer, fc blockers

Which tetramers can be used for my mouse samples?

Posted by Tomohiro Nakajo on Aug 28, 2018 1:00:00 PM

The thymus shapes a host's T-cell repertoire so they do not respond to "self" antigens, yet are able to recognize "non-self" antigen challenges (from pathogens and tumors). T cell participation in these immune responses are dependent on interaction with peptide- and self-MHC. In other words, T cells only recognize the peptides derived from cancers or pathogens if they are embedded in the cleft of self host MHC grooves. Such peptides are processed and presented on the surface of antigen-presenting cells (MHC class I and II in dendritic cells, macrophages, and B cells) or all nucleated cells (MHC class I) allowing T cells to identify infected or cancer cells. Direct detection of such Ag-specific T cells and functional analysis is important for understanding disease immunopathogenesis and the process of drug development. MBL International has had tremendous success in advancing the methodologies to accurately measure antigen-specific T cells. 

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Tags: Tetramer

Quick!  Switch out your peptide! 

Posted by Marc Delcommenne, PhD on Mar 27, 2018 12:58:00 PM

The immunotherapy field is making great progress in aiding many disease treatments.  The hope is that one day, personalized immunotherapy can be used so each patient can use their own immune system to overcome their specific disease affliction.  QuickSwitch is a tool for creating custom MHC class I tetramers quickly and easily.

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Tags: Tetramer, QuickSwitch, Peptide, Peptide Exchange

Quick!  Switch out your peptide!

Posted by Marc Delcommenne, PhD on Jan 20, 2017 11:00:21 AM

The immunotherapy field is making great progress in aiding many disease treatments.  The hope is that one day, personalized immunotherapy can be used so each patient can use their own immune system to overcome their specific disease affliction.  QuickSwitch is a tool for creating custom MHC class I tetramers quickly and easily.

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Tags: Tetramer, QuickSwitch, Peptide, Peptide Exchange

Don't be fooled. Not all tetramers are created equal!

Posted by Bindi M. Doshi, PhD; Cheryl A. Guyre, PhD on Aug 24, 2016 11:45:03 AM

Have you seen comparisons of multimer technology claiming to be superior to tetramers? Be sure to ask which tetramers were used in the comparison! MBL tetramers have a clear advantage over academic tetramers and other commercial MHC multimer products, not only due to the reliability and high quality for every lot produced, but also due to the proprietary alpha-3 mutation. This mutation, engineered into the heavy chain of all of our human and macaque class I tetramers, helps decrease non-specific binding, leading to enhanced specificity. Check out the images below to see the details of this important technology!

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Tags: Tetramer

Help! My mouse tetramer stained ALL the CD8+ T cells!

Posted by Cheryl A. Guyre, Ph.D. on Aug 26, 2015 3:39:00 PM

You’re sitting at your flow cytometer, staring at your CD8 x tetramer plot in anticipation of a tiny but meaningful group of dots appearing in the upper right quadrant. You hold your breath, click “acquire,” and watch… Wait, what? What’s this ugly diagonal?? How come ALL the CD8 positives are dual positive??! Hey, this is my control mouse! That one should be completely tetramer negative! Yep, I’ve been there too. Classic case of using a CD8 antibody that doesn’t play nice with tetramers.

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Tags: Tetramer

Be positive! 5 ways to confirm your MHC tetramer is binding

Posted by Cheryl A. Guyre, Ph.D. on Apr 17, 2015 4:35:17 PM

In the previous Tetramer Tips blog, 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.

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Tags: Tetramer, Immunology

Tetramer Tips for Success: How to be a (Negative) Control Freak

Posted by Cheryl A. Guyre, Ph.D. on Mar 20, 2015 10:48:00 AM

While being a Control Freak may not be the best choice for living in spiritual harmony, it is a downright asset in flow cytometry research!  Controls not only help you set up an experiment to get a clear or “true” answer, they can also help you troubleshoot what may have gone wrong when your data just doesn’t look quite right, so that your next attempt will turn out better.  In flow cytometry, controls are critical to help determine “real” events from artifacts.  So, what should you use as controls in tetramer experiments?

 

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Tags: Tetramer, Immunology

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