Author: codemakers

Product Specification Sheet 1

Posted on by codemakers
Cat. No **Catalog No. inserted here**
Product MHC monomer / MHC Tetramer
MHC allele **Allele inserted here**
Peptide **Peptide inserted here**
Category **Category inserted here**
Conjugate **Dye inserted here**
Size **Size inserted here**
Formulation PBS, pH 7.4 containing, 5% glycerol. A 50 test size contains 250 µL MHC Tetramer reagent. One test is 5 µl. One test contains 0.2 µg MHC monomer conjugated to 0.05 µg streptavidin with relevant fluorochrome.
Recommended use Recommended for detection of antigen-specific T cells by flow cytometry.
Precautions The product is not for clinical use. For research use only (RUO). Please see MSDS.
Storage Store at -20°C if not used the same day. Do not repeatedly freeze/thaw. Can be stored at 4°C while handling.
Approved date **Todays date inserted here**
Background MHC Tetramers recognize CD8+ T cells that are specific for a particular peptide in context of a class I MHC allele. All specific CD8+ T cells regardless of their functional status are detected using MHC Tetramers.
Peptide loading

Procedure for loading MHC specific peptide-antigen into Empty Loadable MHC Tetramers. This procedure does not apply for Off-the-shelf Tetramers or Custom Tetramers.

Materials:

  • Empty-loadable MHC Tetramers.
  • Peptide antigen, 10 mM (or 10 mg/mL) stock solutions (in DMSO).
  • PBS, pH 7.4.

Procedure for loading peptide-antigen into Empty Loadable MHC Tetramers for 1 test (5µL):

  • Prepare a 200 µM working stock of peptide antigen by diluting 10 mM DMSO stock solution in PBS, pH 7.4 (Example: Add 1 µL of 10 mM stock solution to 49 µL PBS). Make sure the final DMSO concentration remain less than 1% when incubating tetramers with cells.
  • Incubate 5 µL of Empty Loadable MHC Tetramers with 0.5 µL of 200µM peptide antigen for 30 minutes on ice/4 °C.
  • Antigen-specific MHC Tetramers can now be used for T-cell staining. Store at -20°C if not used the same day.
  • For staining protocol, please follow our standard procedure for T-cell staining.
Staining procedure Procedure for staining CD8+ T cells with MHC Tetramers.
Avoid bright or direct light due to light-sensitive fluorochromes.Materials:

  • Antigen-specific MHC Tetramers.
  • PBS, PH 7.4.
  • PBS with 2% fetal calf serum (or alternatively 2% BSA).
  • 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).
  • Anti CD3 antibody (Clones SK7, S4.1 and UCHT1 all work well together with the MHC Tetramers).
  • Anti CD4 antibody.

Procedure:

  • Prepare cells: Wash a maximum of 2-10 million lymphoid cells (PBMCs, TILs, 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 MHC Tetramers: MHC Tetramers must be centrifuged at 3000 X g for five minutes prior to incubating with cells.
  • After centrifugation, carefully (without disrupting any pellet) transfer 5 µL of antigen-specific MHC tetramer to cells and mix 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 CD8, anti CD3, 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 MHC Tetramer reagent 10 min. prior to addition of anti-CD8 antibody for optimal Tetramer staining.

 

Product Specification Sheet 2

Posted on by codemakers
Cat. No **Catalog No. inserted here**
Product CD1d Tetramer
MHC allele **Allele inserted here**
Peptide **Peptide inserted here**
Category **Category inserted here**
Conjugate **Dye inserted here**
Size **Size inserted here**
Formulation PBS, pH 7.4 containing, 5% glycerol. A 50 test size contains 250 µL CD1d 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 (RUO). Please see MSDS.
Storage Store at -20°C if not used the same day. Do not repeatedly freeze/thaw. Can be stored at 4°C during handling.
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 at -20°C if not used the same day.
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.