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||MHC Tetramer conjugated with fluorochrome.
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||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 MHC monomer conjugated to 0.05 µg streptavidin with relevant fluorochrome.
||Recommended for detection of antigen-specific T cells by flow cytometry.
||The product is not for clinical use. For research use (RUO) 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.
||Store at -20°C if not used the same day. Do not repeatedly freeze/thaw. Can be stored at 4°C while handling.
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||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.
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.
- 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.
||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.
- 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.