Tips & tricks

General:

Our general staining procedure should be applicable for most T cell staining experiments. However, successful staining depends on an appropriate experimental design. Different MHC alleles and MHC Tetramers carrying specific peptides might have dissimilar staining characteristics. Staining patterns also depend on the type of cell sample, and there will be variations among different donors.

Number of cells to stain:

In general we recommend to stain 2-10 million lymphoid cells (PBMCs, TILs, or splenocytes). However, this may not be enough to see rare events or you may be able to see frequent events in fewer cells. TIL preparations tend to be enriched for antigen-specific T cells and you may detect specific events in as little as 200,000 TILs.

Analysis of results:

MHC Tetramer-positive cells can be viewed by gating on live lymphoid cells and then making a plot showing MHC Tetramer positive cells on the x-axis and anti-CD8 positive cells on the y-axis (anti-CD3 for NKT cells). We always recommend gating on live cells, since dead cells can disturb the staining picture. If cells are less than 80% viable, precautions in interpretation of results should be taken.

Controls:

  • We recommend including a positive control e.g. specific T cell clone/line or PBMCs from a positive donor.
  • We recommend including a negative control e.g. T cell clone/line negative for the used MHC Tetramer or PBMCs from a negative donor.
  • We do not recommend including Empty Loadable MHC Tetramer without peptide as negative control as Empty Loadable MHC Tetramers may give rise to increased background in some donor materials.
  • We recommend including a negative control MHC Tetramer with irrelevant peptide. See below.

 

Cat. No. HLA type Pep. Seq. Antigen
HA02-025 HLA-A*0201 GVAGDVSAV None natural found
HA02-024 HLA-A*0201 AVIAPVHAV None natural found
HA02-014 HLA-A*0201 SLYNTVATL HIV-1 gag p17 76-84

Unspecific staining:

Under certain experimental circumstances, MHC Tetramers can result in unspecific staining. A common source of unspecific staining arises from omitting centrifugation of Tetramers at 3300 x g for five minutes prior to incubating with cells. Additional washing of the cells before and after MHC Tetramer staining can also minimize problems.

 

If unspecific binding remains a problem, then we recommend optimizing incubation times and temperatures and the pH of the incubation and washing buffers. Generally, if the incubation temperature is decreased, the incubation time should be increased and vice versa.

 

Different MHC-peptide combinations can behave differently depending on e.g. cell material and affinity and thus titration of the amount of MHC Tetramer may help solve the problem. Start by testing 2.5 µl, 5 µl, and 10µl of MHC Tetramer.

Anti-CD8/CD3 antibodies:

Some anti-CD8 antibodies can negatively interfere with MHC Tetramer binding to the T cell receptor. The anti-CD8 antibodies Clones RPA-T8 and SK1 recognizing human CD8 and clone KT15 or YTS169.4 recognizing murine CD8 work well in combination with our Tetramers. No anti-CD3 antibodies are known to interfere with our Tetramers.

Compensation:

When using flourochromes with overlapping emission spectra in the same sample always compensate the sample according to the specifications of the flow cytometer. If in doubt about panel design please visit BD Horizon™ Guided Panel Solution.

Fixation:

Fixation of cells is compatible with Tetramer staining but only when fixing samples after Tetramer staining. Do not attempt to fix samples prior to Tetramer staining.
Fix cells for 1-4 hours at r.t. in 1% methanol-free formalin in PBS following staining and washing. Commercial fixing kits can also be used.

Intracellular staining:

Fixation and intracellular staining for cytokines is compatible with MHC Tetramer staining. Tetramer staining should be carried out prior to fixation/permeabilization and staining for intracellular cytokines. Golgi plug (BD bioscience #555029) and Intracellular Fixation & Permeabilization Buffer Set (eBioscience™ #88-8824-00) work well with our MHC Tetramers.