General Tips and Tricks for Tetramer Staining
Our standard staining procedure is suitable for most T cell staining experiments. However, the success of your experiment depends on careful planning. Different MHC alleles and MHC Tetramers with specific peptides may have varying staining characteristics. Staining outcomes also depend on the type of cell sample, and there can be variation between donors.
Number of Cells to Stain
We generally recommend staining 2-10 million lymphoid cells (e.g., PBMCs, TILs, or splenocytes). However, the number of cells needed can vary:
- For rare events, you may need to stain more cells.
- For more frequent events, fewer cells may suffice.
- TILs (Tumor Infiltrating Lymphocytes) tend to be enriched for antigen-specific T cells, and specific events can sometimes be detected with as few as 200,000 TILs.
Analyzing Results
To analyze MHC Tetramer-positive cells, follow these steps:
- Gating: Gate on live lymphoid cells.
- Plotting: Create a plot with MHC Tetramer-positive cells on the x-axis and anti-CD8-positive cells on the y-axis (or anti-CD3 for NKT cells).
- Viability: Always gate on live cells. Dead cells can affect the staining results, and if viability is below 80%, take precautions when interpreting the data.
Controls
We recommend including the following controls in your experiment:
- Positive Control: Use a specific T cell clone/line or PBMCs from a known positive donor.
- Negative Control: Include a T cell clone/line negative for the MHC Tetramer being used, or PBMCs from a negative donor.
Note: We do not recommend using Empty Loadable MHC Tetramers (without peptide) as a negative control, as they may increase background staining in some donor samples. Instead, use an MHC Tetramer with an irrelevant peptide.
Example of irrelevant peptide MHC Tetramers:
Cat. No. | HLA/MHC Type | Peptide Sequence | Antigen | Type |
---|---|---|---|---|
HA24-037 | HLA-A*24:02 | RYAAAAALL | None found in nature | Control |
HA11-021 | HLA-A*11:01 | ATAAAAAAK | None found in nature | Control |
HB07-026 | HLA-B*07:02 | APAAAAAAV | None found in nature | Control |
HA02-179 | HLA-A*02:01 | ALAAAAAAV | None found in nature | Control |
MKb-022 | H-2Kb | ASYAAAAV | None found in nature | Control |
Unspecific Staining
Under certain conditions, MHC Tetramers may cause unspecific staining. To minimize this:
- Centrifuge Tetramers: Always centrifuge Tetramers at 3300 x g for five minutes before incubating with cells.
- Washing: Increase the number of washes before and after staining to reduce background noise.
If unspecific binding persists, try these adjustments:
- Optimize incubation: Adjust incubation times and temperatures. If you lower the incubation temperature, increase the incubation time, and vice versa.
- Titrate Tetramers: Try different amounts of Tetramers (e.g., 2.5 µl, 5 µl, or 10 µl) to find the optimal concentration for your experiment.
Anti-CD8/CD3 Antibodies
Some anti-CD8 antibodies can interfere with MHC Tetramer binding to T cell receptors. The following antibody clones are compatible with our Tetramers:
- Human CD8: Clones RPA-T8 and SK1
- Murine CD8: Clones KT15 and YTS169.4
No anti-CD3 antibodies are known to interfere with our Tetramers.
Compensation
When using fluorochromes with overlapping emission spectra, make sure to compensate your sample properly according to the specifications of your flow cytometer.
Fixation
Fixation of cells is compatible with Tetramer staining but should only be done after Tetramer staining is complete. Follow these steps:
- Fix cells for 1-4 hours at room temperature in 1% methanol-free formalin in PBS after staining and washing.
- Alternatively, you can use commercial fixing kits.
Intracellular Staining
Tetramer staining is compatible with intracellular cytokine staining, but the order of steps is important:
- Tetramer Staining: Perform Tetramer staining before fixation and permeabilization.
- Cytokine Staining: Follow up with intracellular cytokine staining after fixation and permeabilization.
Products such as Golgi Plug (BD Bioscience #555029) and Intracellular Fixation & Permeabilization Buffer Set (eBioscience™ #88-8824-00) work well with our MHC Tetramers.
By following these tips and optimizing your protocol based on your specific experimental conditions, you can improve the accuracy and reliability of your results.