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FAQ

CD1d Tetramer



  1. Can I use DMSO, instead of pyridine, to dissolve α-GalCer?
    We recommend using pyridine. In an evaluation at MBL, no positive cells were detected when α-GalCer was dissolved in DMSO and heated at 80°C for 10 seconds before loading the mouse CD1d Tetramer-PE.
  2. Can I store α-GalCer after dilution to 200 µg/mL?
    Do not store α-GalCer diluted to 200 µg/mL because storage reduces the reactivity of the diluted α-GalCer. Instead, store at −20°C as a 1 mg/mL solution in pyridine, and dilute to 200 µg/mL immediately before loading the CD1d Tetramer.CD1d dilution
  3. Can I store CD1d Tetramer at 4°C after it is loaded with α-GalCer?
    Yes. Both human and mouse CD1d Tetramers remain reactive for at least 12 months.CD1d_load
  4. What is the expected positive rate of CD1d Tetramer?
    Typically, 0.01-0.05% of PBMCs in freshly collected blood from healthy individuals are positive. However, variation between individuals is large, and some individuals may have no positive cells. When performing flow cytometry, we recommend gating on lymphocytes to identify about 100,000 cells for analysis because of the low frequencies. When mouse splenocytes were stained, approximately 5% (BALB/c) and 20% (C57BL/6) of CD4-positive cells were also CD1d Tetramer-positive (test results at MBL).
  5. Can you recommend a blocking agent for use with CD1d Tetramer?
    We recommend Clear Back (Human Fc receptor blocking reagent, MBL Code No. MTG-001).
    It will prevent non-specific uptake of the tetramer reagents by monocyte-derived cells in both humans and mice.
  6. Human and mouse CD1d proteins have a high homology. Do the CD1d Tetramer reagents show species cross-reactivity between human and mouse?
    We recommend matching the reagent to the species from which the cells were derived. Staining human PBMC with mouse CD1d Tetramer is not recommended because of high non-specific staining. Staining mouse splenocytes with human CD1d Tetramer instead of mouse CD1d Tetramer results in a lower percentage of positive cells.CD1d cross-reactivity
  7. There is sometimes non-specific staining when CD3-negative cells are stained with human CD1d tetramer without α-GalCer. What should I do?
    Human CD1d Tetramer without α-GalCer is known to bind to ILT4 (CD85d) protein expressed in CD14-positive cells. CD14-positive cells can be eliminated by gating out after co-staining them with CD14 antibody. For details, please refer to Li D, et al. J. Immunol. 182: 1033-1040 (2009) PubMed: 19124746.