Frequently asked questions

What is a tetramer

A tetramer is a oligomer of four identical monomers. Thus, a MHC tetramer is four identical peptide-MHC monomer molecules assembled by a (fluorochrome-conjugated) streptavidin molecule. 

MHC class I Tetramers can be used to label CD8+ T cell subsets expressing a cognate T cell Receptor (TcR) which will interact with a specific peptide-MHC complex (pMHC). 

Similarly, will MHC class II Tetramers interact with the CD4+ T cell subsets expressing a TcR specific to the peptide-MHC class II complex. 

The pMHC:TcR interaction is weak and fast dissociating, which prevents a distinct labelling of T cells. Tetramerization of pMHC monomers increases the avidity resulting in a stable interaction between the tetrameric pMHC and specific TcRs which can be detected in a flow cytometer. Identification of antigen-specific T cell was first published by Altman at Standford in 1996

What is a tetramer staining?

A peptide MHC Tetramer staining is a functional assay where antigen-specific T cells are stained with e.g. a fluorochrome-labelled MHC tetramer loaded with the corresponding peptide epitope. A flow cytometry analysis allows you to detect and monitor antigen-specific T cells — expressing the cognate T cell receptor to the peptide MHC complex in question —  in a population of T cells with multiple specificities. Co-staining with cell surface markers or intracellular staining staining for cytokines and chemokines, can provide you with a detailed view and profile of the antigen-specific T cells of interest. 

MHC tetramers allows:

  • Direct detection and monitoring of antigen-specific T cells
  • Different fluorochrome-conjugations allows simultaneously detection of multiple T cell specificities 
  • Combined staining with other cell surface markers or intracellular cytokine or chemokine staining
  • Staining of PBMCs from blood samples, tissue-derived cell samples or in vitro cell cultures 
  • High-throughput of large number of cell samples
  • FACS sorting of MHC tetramer labelled T cells for downstream functional assays and analysis

What is an easYmer®

easYmers® are basically “empty” HLA class I molecules. Our proprietary formulation renders highly active peptide receptive HLA class I molecules which allows any non-expert user to, seamlessly, produce HLA monomers and tetramers just by adding peptides of interest. 

Included in the easYmer kit is an allotype relevant postive control peptide, which allows you to validate the quality of your own peptide-HLA complexes in a simple flow cytometry based assay. 

Read more about easYmers® here 

Do I need special equipment to setup an easYmer® complex?

No, there are no special equipment requirements to set up an easYmer folding. 

What are the advantages of easYmers®

The HLA easYmers provides maximum flexibility for your T cell research compared to premade or even custom HLA tetramers. 

  • The easYmers allows you to maintain a repository of “empty” HLA molecules in your own lab allowing you to make your own custom mono- or tetramers from day-to-day similar to what you would only find in a tetramer core facility service
  • The easYmers are stable for >18 months at -20°C in its “empty” peptide receptive conformation.
  • All easYmers are 100% biotinylated providing maximum flexibility in terms of choosing flow colours or metal-chelated streptavidin
  • Use the easYmers to screen a large number of different epitopes or make a large amount of a single HLA class I tetramer and screen multiple donors. 
  • You are no longer limited to work on the most common available allotypes – the easYmers are available in more than 30 allotypes (HLA-A, HLA-B and HLA-C). See our full range of available allotypes here 
  • You can use our flow cytometry based protocols to asses the folding efficiency of your custom peptide HLA complexes
  • Keep your epitope sequence proprietary as there is no need to disclose the sequence to a custom tetramer manufactor

How long can I store the easYmer®

The easYmers® are supplied in a stabilized peptide receptive conformation which allows storing in the freezer (-20°C) for 24 months. 

Once a peptide have been loaded the resulting peptide-HLA monomer is stable in the freezer (-20°C) for years. 

How, and how long can I store Monomers and Tetramers?

Peptide-MHC class I / class II monomers are stable in the freezer (-20°C) for years — however, we recommend to avoid repeated freeze-thaw cycles. All our molecules are biotinylated, so it is possible to store the monomer for years and just assemble the tetramers at will. 

Tetramers (four peptide-MHC monomers assembled by streptavidin) should be stored in the fridge (4°C) where it is stable for months. The long-term stability of tetramers may depend on the peptide, thus some tetramers are stable for longer periods of time than others

Can I store Tetramers in the freezer?

We do not recommend to store a fluorescence-conjugated Tetramer frozen (below 0°C). If you need to store a Tetramer for a longer period of time, we recommend storing it in the fridge (4°C).

Do you perform QC of your MHC monomers and tetramers?

All our catalog and custom HLA monomers are being functional tested before tetramerization and shipping. 

Each lot of our easYmers® undergoes functionally testing with positive control peptides to validate that the folding efficiency 

Can I fixate T cell prior to Tetramer staining?

No, to get optimal tetramer staining of your antigen-specific T cells you should work with live cells. 

You can however, fixate the tetramer stained cells in PBS suppl. with 1% formaldehyde if you wish to analyse your cells at a later time. 

How do I identify the optimal peptide

MHC class I molecules typically binds nonamer (9-mer) peptides but other peptide lengths may be relevant. Especially for MHC class I it is important to identify the optimal peptide you wish to bind to the MHC molecule of interest.  We recommend using a MHC binding prediction tool before making tetramers with easYmers® or ordering a tetramer:

NetMHC 4.0 Server or NetMHCpan 4.0 Server