Protocols

1. Procedure for loading Empty Loadable MHC Tetramers with peptide antigen

This procedure successfully loads MHC specific peptide-antigen into empty MHC tetramers in a single step. Do not apply UV exposure, chaotropic salts, heating or other means. Just add peptide. All of our Empty Loadable MHC Tetramers are available with our full range of fluorophore conjugates.

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 the cells.
  • Incubate 5 µL of Empty Loadable MHC Tetramers with 0.5 µL of peptide antigen (200 µM) for 30 minutes on ice/4 °C.
  • Antigen-specific MHC Tetramers can now be used for T-cell staining. Keep at 4 °C until further use.
  • Use 5 µL of antigen-loaded Tetramers for each staining.
  • For staining protocol, please follow our below described standard procedure for T-cell staining.

 

 

Empty loadable MHC Tetramers. Just add peptide and instantly stain T cell samples.

 

2. Procedure for staining T cells with MHC Tetramers

Avoid exposure of MHC tetramers to 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 (none known to interfere with tetramer staining).
  • Anti CD4 antibody.

Procedure:

  • Prepare cells: Wash 2-10 x 106 lymphoid cells (PBMCs, TILs, or splenocytes) with PBS containing 2% fetal calf serum in appropriate volume (200 µL if using a 96 well plate or 12 x 75 mm polystyrene test tubes) and centrifuge at 300 x g for 5 minutes.
  • Discard the supernatant and dissolve the cell pellet with 50 µL PBS.
  • Prepare MHC tetramers: Antigen specific MHC tetramers must be centrifuged at 3300 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 the reaction for 15 minutes at 37°C in the dark.
  • Post incubation; add an optimal amount of cell surface marker antibodies conjugated with appropriate fluorophores in 20 µL PBS with 2% fetal calf serum. For best separation of desired cell population, suggested cell surface markers are; anti CD8, anti CD3, anti CD4 and a live/dead marker.
  • Resuspend and incubate for 30 minutes at 4 °C in dark.
  • Wash the cells twice with an appropriate volume of PBS containing 2% fetal calf serum followed by centrifugation at 300 x g for 5 minutes.
  • Transfer the samples to FACS tubes and analyze on flow cytometer.

 

It is important that cells be incubated with the MHC Tetramer reagent 15 min. prior to addition of anti-CD8 antibody. If anti-CD8 antibody is included before or simultaneously with MHC Tetramer, a poorer resolution of positive and negative cell populations may result.

 

MHC Tetramer Reagents are for research use only. Not for use in diagnostic procedures

 

 

3. Procedure for dual staining of T cells with MHC Tetramers

Avoid exposure of MHC tetramers to bright or direct light due to light-sensitive fluorochromes.

 

Purpose: Staining of T cells in lymphoid cell preparations with MHC Tetramers of same specificity conjugated with two different fluorophores in the same sample. This procedure can improve the detection of rare T cells or T cells with low fluorescence intensity.

 

Materials:

  • Antigen‑specific MHC Tetramers with fluorochrome conjugate 1 (e.g. PE)
  • Antigen‑specific MHC Tetramers with fluorochrome conjugate 2 (e.g. APC)
  • 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 (none known to interfere with tetramer staining).
  • Anti‑CD4 antibody

Procedure:

  • Prepare cells: Wash 2-10 x 106 lymphoid cells (PBMCs, TILs, or splenocytes) with PBS containing 2% fetal calf serum in appropriate volume (200 µL if using a 96 well plate or 1 mL if using 12×75 mm polystyrene test tubes) and centrifuge at 300 X g for 5 minutes.
  • Discard the supernatant and dissolve the cell pellet with 40 µL PBS.
  • Prepare MHC tetramers: Antigen‑specific MHC tetramers must be centrifuged at 3300 x g for five minutes prior to incubating with cells.
  • After centrifugation, (without disrupting any pellet) transfer 2,5 µL of each of the two differently fluorochrome labeled antigen‑specific MHC tetramers to the cells and mix gently.
  • Incubate the reaction for 15 minutes at 37°C in dark.
  • Post incubation; add an optimal amount of cell surface marker antibodies conjugated with appropriate fluorophores in 20 µL PBS with 2% fetal calf serum. For best separation of desired cell population, suggested cell surface markers are; anti CD8, anti CD3, anti CD4 and a live/dead marker. Resuspend and incubate for 30 minutes at 4°C in dark.
  • Wash the cells twice with PBS containing 2% fetal calf serum followed by centrifugation at 300 X g for 5 minutes. Transfer the samples to FACS tubes and analyze on flow cytometer.

 

It is important that cells be incubated with the MHC Tetramer reagents 10 min prior to addition of anti-CD8 antibody. If anti-CD8 antibody is included before or simultaneously with MHC Tetramer, a poorer resolution of positive and negative cell populations may result.

 

MHC Tetramer Reagents are for research use only. Not for use in diagnostic procedures.

 

 

4. Procedure for loading empty CD1d Tetramers with lipid antigen

This procedure successfully loads Alpha-Galactosyl Ceramide into ligand-empty human and mouse CD1d tetramers. CD1d monomer is incubated with 15 molar excess of lipid antigen during loading. Optimal conditions and molar stoichiometry will need optimization for loading other lipid antigens.

Materials:

  • Lipid antigen (e.g. Alpha-GalCer from S&D Lipopharma) stock solution dissolved in DMSO at 1 mg/ml (1.2 mM).
  • 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. Heat it to 80 °C for 1 min to completely dissolve the lipid. Cool the lipid solution to 37 °C.
  • Add 2.5 μl of the dissolved lipid antigen to 50 μl of empty CD1d tetramer tempered to 37 °C.
    Incubate at 37 °C for four hours in the dark.
  • Store the lipid loaded CD1d Tetramer at 4 °C in the dark until use.

 

5. Procedure for staining NKT cells with CD1d Tetramers

Materials:

  • Antigen specific CD1d Tetramers
    PBS, PH 7.4
    PBS with 2% fetal calf serum (or alternatively 2% BSA).
  • Anti CD3 antibody (none known to interfere with tetramer staining).
  • 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:

Avoid exposure of CD1d tetramers to bright or direct light due to light-sensitive fluorochromes.

 

  • Transfer 2-10 million lymphoid cells (PBMC or splenocytes) to a FACS tube. Allocate less cells when staining NKT cell clones.
  • Wash cells by adding 1 mL PBS containing 2% fetal calf serum. Centrifuge at 300 x g for 5 min. Remove supernatant and resuspend cells in a total volume of 50 µL PBS containing 2% fetal calf serum.
  • Add 5 µL CD1d tetramer and mix gently. Incubate in the dark at room temperature for 10 min.
  • Add an optimal amount of anti-CD3 antibody conjugated with a relevant flourochrome. Additional antibodies (e.g. anti-CD8 and anti-CD4) conjugated with relevant fluorochromes as well as live/dead markers may also be added at this step. Incubate in the dark at r.t. for 20 min.
  • Wash cells by adding 2 mL PBS containing 2% fetal calf serum. Centrifuge at 300 x g for 5 min. and remove the supernatant.
  • Repeat washing step.
  • Resuspend cells in 200 µL PBS. Analyze on a flow cytometer or store cold in the dark until analysis. Do not store longer than two hours before analysis for optimal results.

CD1d tetramers are for research use only. Not for use in diagnostic procedures.