Tips & Tricks
MHC Dextramers are designed to detect rare occurrence of antigen-specific T cells. However, a successful staining depends on an appropriate experimental design. The General Staining Procedure should be applicable for most experiments if the MHC Dextramers are handled according to their specifications and the flow cytometer has been set up properly (see Flow Cytometer Set-Up). It should be noted that different MHC alleles and MHC Dextramers carrying distinct peptides could have dissimilar staining characteristics. One of the reasons for this is that they have various affinities for the T-cell receptor. Staining patterns also depend on the type of cell sample and there will be variations among different donors.
The notes below should help in optimizing experiments with a particular MHC Dextramer™.
Analysis of Results:
The MHC-Dextramer-positive cells are most easily viewed by gating on live lymphoid
cells (see Flow Cytometer Set-Up) and then making a plot showing MHC Dextramer™ on
the X-axis and anti-CD8 on the Y-axis. It is very important to gate on live cells,
as dead cells can obscure the staining picture. The viability of the cell sample should preferably be > 80%.
Controls:
Include a positive control if possible (e.g. specific T-cell clone/line, PBMC
from a positive donor) and a negative control (T-cell clone/line negative for the
used MHC Dextramer™, PBMC from a negative donor, MHC Dextramer™ with another allele
and/or irrelevant peptide).
Speeding Up the Staining Procedure:
MHC Dextramers and conjugated anti-CD8 antibody can be mixed prior to addition
to the lymphoid cells, then incubated at 4 şC for 20 minutes followed by wash, etc.
Not all anti-CD8 antibodies can be used in this type of staining procedure. Clone
DK25 for human cells and clones KT15 or YTS169.4 for murine cells have been
shown to work well together with the MHC dextramers. Clone 53-6.7
cannot be used in this type of set-up. It should be noted that mixing MHC Dextramer™
and conjugated anti-CD8 antibody prior to addition to the cells can result in a
smaller signal-to-noise-ratio compared to results obtained following the General
Staining Procedure.
Nonspecific Staining:
Under certain conditions, MHC Dextramers can give rise to nonspecific staining.
This phenomenon is most commonly observed with the MHC/FITC Dextramers. An extra wash
before and after MHC Dextramer™ staining can minimize the
problem. If nonspecific binding seems to be a problem, it is recommended to compare
different incubation times and temperatures or change pH of the incubation and washing
buffers. Generally, if the incubation temperature is lowered, increase the incubation
time, and if the incubation temperature is increased, shorten the incubation time.
Even though 10 µL of MHC Dextramer™ is recommended per staining, various alleles
can behave differently depending on e.g. cell material or affinity. Titration of
the MHC Dextramers may help solve the problem.
Anti-CD8/CD3 Antibodies:
Some anti-CD8 and anti-CD3 antibodies can interfere with MHC Dextramer™ binding.
Be sure to use an antibody not interfering with MHC Dextramer™ binding. Titrate the
anti-CD8 or anti-CD3 antibody before use. Anti-CD8 clones DK25, KT15 and YTS169.4
and anti-CD3 clones UCHT1, C363.29B and KT3 works well together with MHC Dextramers.
Compensation:
When using flourochromes with overlapping emission spectra,
remember to compensate the sample according to the specifications of the flow cytometer.
Fixation:
It is recommended to wash samples prior to fixation. Fixation should be carried
out after staining with MHC Dextramers. Pre-staining fixation is not recommended.
MHC Dextramers work well with fixing and lysing reagents such as Uti-Lyse™ Erythrocyte-Lysing Reagent (Dako Codes S3325/S3350). Uti-Lyse™ lyses erythrocytes and fixes and stabilizes leucocytes in whole blood. Fixation and intracellular staining with Dako IntraStain, Fixation and Permeabilization Kit for Flow Cytometry (Dako Code K2311) can be used together with MHC Dextramers. Additional washing step prior to IntraStain Reagent A is essential for good results using MHC Dextramer™ together with the IntraStain procedure.
For research use only. Not for use in diagnostic procedures.
Products and support
If you need information concerning Immudex products, have a technical enquiry, or are interested in customer-defined Dextramers and/or collaboration with Immudex, please contact us on Email customer@immudex.com
If you need ordering information, please contact on us Email ordering@immudex.com
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