Tips and Tricks

Improve Your Experiment With Dextramer® 

A successful staining with Dextramer depends on the appropriate experimental design. General staining procedures apply to most of the experiments. However, different Dextramer reagents can have dissimilar staining characteristics, depending on the MHC-peptide complex displayed.

Please find a collection of various Tips and Tricks to optimize an experiment with Dextramer here: 

Controls

Positive and negative controls should be included in the experiment with MHC Dextramer reagents.

Examples of positive controls:

  • staining of antigen specific T-cell clone or T-cell line, or PBMC from a positive donor

Examples of negative controls:

  • staining of a T-cell clone or T-cell line negative for the MHC Dextramer, or PBMC from a negative donor
  • MHC Dextramer carrying MHC alleles unmatched with the sample
  • MHC Dextramer carrying peptides unrelated with the experimental setup for which a positive response is not expected.

Speeding Up the Staining Procedure

MHC Dextramer and anti-CD8 antibody can be mixed and added simultaneously to the lymphoid cells, then incubated at 4°C for 20 minutes followed by washing steps (step 5-7 in the General staining protocol). Not all anti-CD8 antibody clones are however suitable for this staining procedure.  In our experience, the anti-CD8 antibody clones SK1 (BD) and KT15 or YTS169.4 (Dako) can be used for this particular procedure, whereas the clone 53-6.7 (BD) cannot.

It should be noted that mixing MHC Dextramer and anti-CD8 antibody before staining can result in a smaller signal-to-noise ratio compared to general staining procedure.

Anti-CD8/CD3 Antibodies

Some anti-CD8 and anti-CD3 antibodies can interfere with Dextramer binding.

We recommend to titrate the anti-CD8 and/or anti-CD3 antibody before use to assess whether the antibody interferes with Dextramer binding.

Dextramer reagents are compatible with:

  • anti human CD8 antibody clone SK1 (from BD)
  • anti murine CD8 antibody clones KT15/ YTS169.4 (from Dako)
  • anti human CD3 antibody clones SK7 (BD, eBiosciences)/ S4.1 (Invitrogen)/ UCHT1 (Dako) 

Unspecific Staining

Some MHC-peptide combinations can cause higher unspecific staining with MHC Dextramer®. In this case, additional washing of the cells before and after MHC Dextramer staining can minimize the unspecific binding.

If the problem is not solved by increased washing, it is recommended to compare different incubation times and temperatures. Generally, if the incubation temperature is lowered, the incubation time is increased, and if the incubation temperature is increased, the incubation time is shortened.

Generally, we recommend to use 10 µl of MHC Dextramer to stain up to 1-3 x 10^6 lymphoid cells or 1-3 x 10^5 clonal antigen specific T-cells. However, for some MHC-peptide combinations, a titration of the MHC Dextramer reagent might be advised.

Intracellular Staining

Fixation and intracellular staining with Dako IntraStain and Fixation and Permeabilization Kit for Flow Cytometry can be used together with MHC Dextramer. Additional washing step prior to IntraStain Reagent A is essential for good results, using MHC Dextramer

Fixation

It is recommended to wash samples prior to fixation. Fixation should be carried out after staining with MHC Dextramer. Pre-staining fixation is not recommended.

2% formalin (methanol-free) in PBS can be used as fixative, following staining and washing. MHC Dextramer is compatible with reagents for fixation and lysing such as Uti-Lyse™ Erythrocyte-Lysing Reagent (Dako). 

Analysis of Flow Cytometry Data

To visualize MHC Dextramer positive cells, it is recommended to create a gate on live lymphoid cells and then to make a plot, showing MHC Dextramer on the x-axis and anti-CD8 antibody on y-axis. It is very important to gate on live cells, as dead cells can obscure the staining picture. Cell viability should be > 80%.

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