Product Specification Sheet 2

Cat. No **Catalog No. inserted here**
Product supplied CD1d Tetramer conjugated with fluorochrome.
MHC allele **Allele inserted here**
Peptide **Peptide inserted here**
Category **Category inserted here**
Dye/fluorochrome **Dye inserted here**
Size **Size inserted here**
Formulation PBS, pH 7.4 containing, 0,5% BSA, 5% glycerol and < 0.01% NaN3. A 50 test size contains 250 µL MHC Tetramer reagent. One test is 5 µl. One test contains 0.2 µg CD1d monomer conjugated to 0.05 µg streptavidin with relevant fluorochrome.
Recommended use Recommended for detection of NKT cells by flow cytometry.
Precautions The product is not for clinical use. For research use only.
The product contains sodium azide (NaN3). At product concentrations, though not classified as hazardous, sodium azide may react with lead and copper plumbing to form highly explosive build-ups of metal azides. Upon disposal, flush with large volumes of water to prevent metal azide build-up in plumbing. Please see MSDS.
Storage Keep at 2-8°C if used for NKT cell staining within a week. Store at -20°C for long-term storage. Do not repeatedly freeze/thaw.
Approved date **Todays date inserted here**
Background CD1d Tetramers recognize NKT cells that are specific for a particular lipid antigen in context of CD1d. All specific NKT cells regardless of their functional status are detected using CD1d Tetramers.
Lipid loading This procedure successfully loads Alpha-Galactosyl Ceramide into ligand-empty human and mouse CD1d Tetramers. Optimal conditions and molar stoichiometry will need optimization for loading other lipid antigens.
This procedure does not apply for CD1d Tetramers already loaded with Alpha-GalCer.

Materials:

  • Lipid antigen (e.g. Alpha-GalCer) stock solution dissolved in DMSO at 1 mg/ml (1.2 mM) (http://sdlipopharma.com/products/products.html)
  • Empty CD1d Tetramer
  • 0.5 % Tween-20 in PBS

Procedure for lipid loading of 10 tests of empty CD1d tetramers:

  • Heat the lipid antigen stock solution to 80°C for 1 min to completely dissolve the lipid in DMSO. Dilute lipid antigen five fold into 0.5 % Tween-20 in PBS, to a final concentration of 0.2 mg/ml.
  • Add 5µl of the dissolved lipid antigen to 50 µl of empty CD1d Tetramer.
  • Incubate at 37°C for four hours in the dark.
  • Store the lipid loaded CD1d Tetramer at 4°C in the dark until use.
Staining procedure Procedure for staining NKT cells with CD1d Tetramers.
Avoid bright or direct light due to light-sensitive fluorochromes.

Materials:

  • Lipid loaded CD1d Tetramers.
  • PBS, PH 7.4.
  • PBS with 2% fetal calf serum.
  • Anti CD3 antibody (Clones SK7, S4.1 and UCHT1 all work well together with the CD1d Tetramers).
  • Optionally anti CD8 antibody (Clones RPA-T8 and SK1 recognizing human CD8 and clone KT15 or YTS169.4 recognizing murine CD8 work well in combination with our Tetramers. Some other clones are known to interfere with Tetramer staining).
  • Optionally anti CD4 antibody.

Procedure:

  • Prepare cells: Wash a maximum of 2-10 million lymphoid cells (PBMCs or splenocytes) with PBS containing 2% fetal calf serum in an appropriate volume (use 96 well plate or 12 x 75 mm polystyrene test tubes) and centrifuge at 300Xg for 5 minutes.
  • Discard the supernatant and dissolve the cell pellet in 50 µL PBS.
  • Prepare CD1d Tetramers: CD1d Tetramers must be centrifuged at 3000 X g for five minutes prior to incubating with cells.
  • Add 5 µL of CD1d tetramer for each staining and mix cells gently.
  • Incubate for 15 minutes at 37°C in the dark.
  • Now add an optimal amount of cell surface marker antibodies conjugated with appropriate fluorochromes in a 20-µL volume. Suggested cell surface markers are anti CD3 and optionally anti CD8, anti CD4, and a live/dead marker.
  • Resuspend cells and incubate for 30 minutes at 4°C in dark.
  • Wash cells twice with an appropriate volume of PBS containing 2% fetal calf serum and collect cells by centrifugation at 300Xg for 5 minutes.
  • Transfer cells to FACS tubes and analyze on flow cytometer.

It is important that cells are incubated with CD1d Tetramer reagent 10 min. prior to addition of anti-CD8 antibody for optimal Tetramer staining.

 

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