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HOME > FAQ > 【MHC Tetramer】Optimization/Troubleshooting


【MHC Tetramer】Optimization/Troubleshooting

  1. What is the recommended cell labeling procedure?
    Protocols for whole blood and mononuclear cell preparations are included on the datasheet that accompanies the product. Datasheets can also be found on the MBL website.
  2. What lyse and fixative reagents are recommended?
    Commercial red blood cell lysing reagents such as OptiLyse C (Beckman Coulter, Code No. A11895) or OptiLyse B (BD Biosciences, Code No. IM-1400) have been shown to be compatible with Tetramer whole blood staining protocols. It is important to read the manufacturer’s recommendations with such reagents to be certain that a suitable anticoagulant is used during blood collection. Methanol-free formaldehyde fixative solutions (e.g. PBS containing 0.5% paraformaldehyde or formalin) can be used with Tetramers, as indicated in the tetramer staining protocols. Please be sure to analyze it within 24 hours. Fixing cells prior to tetramer staining is not recommended, as some Tetramers will not bind post-fixation.
  3. Can I stain with Tetramer and antibodies simultaneously?
    Especially for mouse MHC class I Tetramer staining, staining with tetramer and antibodies simultaneously may cause non-specific binding or inhibition of binding to TCR. As described in a datasheet, sequential addition (Tetramer first, antibody second) is recommended to obtain better results.
  4. What Tetramer should I use as a negative control?
    For MHC class I Tetramers, Tetramers loaded with artificially designed peptides that do not exist in nature can be used as negative controls. Because there are few people with HIV in Japan, Tetramers loaded with HIV peptides can also be used as a negative control.
    For MHC class II Tetramers, CLIP peptide loaded tetramer products can be used as a negative control. Because MHC class II molecule loaded with CLIP peptides are formed during the process of MHC class II being presented on the cell surface, CLIP-specific T cells do not exist in nature. If you cannot find any Negative Tetramers of your desired allele described above, a tetramer loaded with a peptide unrelated to your experimental system can also be used as a control.
  5. What can be done about high background staining?
    MHC Tetramer labeling may need to be optimized in your particular system. Tetramer staining best practices may include any or all of the following:
    • Proper titration of Abs/multicolor panel optimization (PMT balance, minimal spillover, etc.)
    • Proper staining temperature: for 30-60 min. at 2-8°C
    • Use of a singlet gate on the flow cytometer to eliminate doublets and aggregates
    • Use of viability dyes (e.g. 7-AAD), particularly when using thawed cells
    • Use of dump channels for exclusion of unwanted cell types (B, NK, monocytes, etc.)
    • Use of positive gating (e.g. CD3)
    • Use an Fc receptor block (e.g. Clear Back: Code No. MTG-001) when appropriate
  6. Which CD8 clone should I use?
    Anti-human CD8 clone Hit8a or SFCI21Thy2D3 (“T8”), and anti-mouse CD8 clone KT15 are recommended. We checked the effects of 10 anti-CD8 antibody clones on MHC Tetramer staining. Please find details in the application note.
  7. Do CD8 antibodies affect the binding of tetramer to specific CD8 T cells?
    The α3 mutation that significantly reduces the binding of MHC Tetramer to human and monkey CD8 also minimizes the aberrant effect that some CD8 antibodies have on the specific binding of MHC class I Tetramer. Therefore, many different anti-human CD8 antibody clones are compatible with Tetramers. Clone Hit8a or SFCI21Thy2D3 (also known as “T8”) are recommended. For experiments on murine cells, the CD8 clone choice is more critical in that clone 53-6.7 should be avoided if possible. Clone KT15 is strongly recommended.
  8. Do CD4 antibodies affect the binding of Tetramer to specific CD4 T cells?
    The choice of anti-CD4 antibody clone is also important like anti-CD8 antibody (Please refer Q.7) because MHC class II has binding site to CD4 molecule. It is reported in the publication (Wooldridge L et al. Eur J Immunol. 2006, 36, 1847-1855).
  9. How do I reduce background in my mouse Tetramer experiment?
    The general method of optimization is described in A.5. Especially, anti-mouse CD8 clone choice is critical when using mouse Tetramers, as some clones, including 53-6.7, lead to high background staining of all CD8+ T cells, rather than the antigen-specific ones. For best results, clone KT15, available from MBL in PE (Code No. D271-5), FITC (Code No. D271-4) and Alexa Fluor® 647 (Code No. D271-A64), is recommended. Additional information can be found in the datasheet that accompanies mouse class I Tetramers.
  10. Which murine allele should be used with C57BL/6 or BALB/c mice?
    Please check mouse strain H-2K, H-2D, H-2L (MHC class I), and I-A (MHC class II) haplotypes below.
    H-2K allele H-2Kb H-2Kd H-2Kk
    Mouse strains C57BL/-, BXSB/Mp, 129/- BALB/c, DBA/2, B10D2, NOD C3H/He

    H-2D allele H-2Db H-2Dd H-2Dk
    Mouse strains C57BL/-, BXSB/Mp, 129/-, NOD BALB/c, DBA/2 C3H/He

    H-2L allele H-2Ld H-2Lq
    Mouse strains BALB/c, DBA/2, B10D2 DBA/1, SWR/J

    I-A allele I-Ab I-Ad I-Ak I-AS
    Mouse strains C57BL/-, BXSB/Mp, 129/- BALB/c, DBA/2 C3H/He SJL/J, B10.S
  11. Which blood collection tubes are compatible with tetramers?
    EDTA, Lithium Heparin, and Sodium Heparin tubes are all compatible with the tetramer staining protocol. However, some of these anticoagulants (e.g. Lithium Heparin) are NOT compatible with particular lysing buffers. Be sure to check the manufacturer’s recommended protocols for reagent compatibility.
  12. Can I use a biotinylated antibody as part of my staining protocol?
    It is not recommended to use biotinylated antibodies and streptavidin conjugates in parallel with MHC Tetramers and particularly with biotinylated Monomers as they could cause non specific staining reactions.
  13. Can tetramers be used to stain tissue sections?
    Yes, tetramer staining of tissue sections has been described in the literature:

    Kawabata T et al. J Med Virol 84, 1120-1127 (2012) (PMID: 22585731)(MBL Tetramer is used)
    Coppieters KT et al. J Exp Med 209, 51-60 (2012) (PMID: 22213807)
    Hong JJ et al. PloS one 4, e4131 (2009) (PMID: 19122815)
    Kuon W et al. J Immunol 173, 4859-4866 (2004) (PMID: 15470026)
    Skinner PJ et al. J Immunol 165, 613-617 (2000) (PMID: 10878330)