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.

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 5 minutes at rt or 30 minutes on ice/4 °C.
  • Antigen-specific MHC Tetramers can now be used for T-cell staining. Keep at 4°C if used same day or store at minus 20°C. Do not repeatedly freeze/thaw.
  • 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 with 2% fetal calf serum.
  • 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 30-60 minutes at 4°C or 10-15 minutes at 37°C, in the dark. Incubation times and temperatures can be optimized according to your specific procedures.
  • 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 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 with 2% fetal calf serum.
  • 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 30-60 minutes at 4°C or 10-15 minutes at 37°C, in the dark. Incubation times and temperatures can be optimized according to your specific procedures.
  • 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 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.

If attempting to load CD1d monomer with Alpha-Galactosyl Ceramide this should be incubated with at least 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.

 

  • 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 with 2% fetal calf serum.
  • 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 30-60 minutes at 4°C or 10-15 minutes at 37°C, in the dark. Incubation times and temperatures can be optimized according to your specific procedures.
  • 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 prior to addition of anti-CD8 antibody for optimal Tetramer staining.

 

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

 

6. Procedure for making MHC tetramers from biotinylated MHC monomers (one-pot method)

Short summary: assemble fluorescent streptavidin (SA) conjugate with peptide-loaded, biotinylated MHC monomer at an approximate 1 SA : 4 MHC molar ratio (one SA has 4 biotin binding sites). Work in PBS (optionally 5% glycerol / 0.5% BSA), add components in the order shown, incubate on ice ~30 min, then use for staining.

Reagents & materials

  • Biotinylated MHC monomer (peptide-empty or preloaded as required).

  • Streptavidin–fluorophore conjugate (azide-free if you will use live cells). Common vendors: BD, BioLegend, others — any azide-free SA conjugate with a known concentration works.

  • Peptide of interest (crude OK if mass verified by MS).

  • PBS (pH ~7.2–7.4).

  • Optional stabilizers: 5% glycerol and 0.5% BSA (good for long-term/storage; omit BSA if it interferes with downstream assays).

  • DMSO (for dissolving peptide).

  • Low-binding tubes, ice, pipettes.

Principle / Ratios

  • The molecular principle: one streptavidin molecule can bind up to four biotinylated MHC monomers, so aim for one SA : four MHC (molar basis) to produce tetramers predominantly.

  • In practice we use a 4:1 mass ratio (MHC : SA) when using typical MHC monomer and SA conjugate preparations — this works since the MW of both MHC monomers and SA is approximately 50KDa.

Peptide preparation

  1. Dissolve peptide in DMSO to a high stock (example: 10 mg/mL).

    • For a 1000 Da peptide, 10 mg/mL ≈ 10 mM. (Check exact MW per peptide.)

  2. Dilute stock into PBS to make a working peptide solution (e.g., 200 µM). Always keep peptide in excess relative to MHC to drive loading. A common final peptide concentration during assembly is ~10–50 µM; we use 20 µM final as a good starting point.

Monomer & streptavidin prep

  • We supply biotinylated MHC monomer at a convenient working concentration 1 mg/mL (1 µg/µL).

  • Use streptavidin–fluorophore at its supplied concentration (note the manufacturer’s mg/mL). If the SA prep contains preservatives (e.g., sodium azide) avoid it for live cell stains.

One-pot tetramer assembly — stepwise (general)

  1. Label a low-binding tube and chill on ice. Prepare final reaction volume based on how many tests you need. (See example below.)

  2. To the tube, add PBS (or PBS + 5% glycerol + 0.5% BSA) to bring the desired final volume.

  3. Add streptavidin–fluorophore (make sure you know its concentration). Mix gently.

  4. Add peptide working solution (so peptide is present in excess). Mix by gentle pipetting.

  5. Add biotinylated MHC monomer last. Mix gently but thoroughly.

    • Rationale for order: adding peptide before monomer ensures peptide is present to load newly added MHC.

  6. Incubate on ice for ~30 minutes. Gently invert or flick the tube once or twice during incubation.

  7. After incubation, use tetramer directly for staining or aliquot and store (see storage notes).

Example

Recipe to make 250 µL tetramer mix which yields 50 tests at 5 µL/test. Here’s the general example converted and explained so it’s easy to adapt:

  • PBS: 202.5 µL

  • Streptavidin conjugate: 12.5 µL of 0.2 mg/mL (this is 0.2 µg/µL)

  • Peptide: 25 µL of 200 µM

  • MHC monomer (biotinylated): 10 µL of 1 µg/µL (i.e., 1 mg/mL)

  • Final mix volume: 250 µL

Arithmetic checks (step-by-step):

  • Streptavidin mass added = 12.5 µL × 0.2 µg/µL = 2.5 µg SA.

  • MHC monomer mass added = 10 µL × 1 µg/µL = 10 µg MHC.

  • Mass ratio MHC : SA = 10 µg : 2.5 µg = 4 : 1

  • Per test (5 µL/test): MHC per test = (10 µg / 250 µL) × 5 µL = 0.2 µg MHC/test. SA per test = (2.5 µg / 250 µL) × 5 µL = 0.05 µg SA/test.

  • Peptide final concentration: 25 µL of 200 µM into 250 µL total → (25/250)×200 µM = 20 µM final peptide.

Use this template but substitute concentrations/volumes according to your monomer and streptavidin stock concentrations. If your SA concentration is different, change the SA volume so that molar (or mass, if you prefer) ratio remains ~1 SA : 4 MHC.

Storage & stability

  • Fresh tetramer: good for immediate staining after 30 min on ice.

  • Short term: store at 4 °C and use within days–weeks.

  • Long term: aliquot (single-use volumes) and freeze (−20 °C) for months–years. Avoid multiple freeze–thaw cycles.

  • Always protect fluorophore from light.