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Quality
Control
All tetramers have been purified by size exclusion and ion exchange chromatography.
Furthermore, each tetramer is tested for the level of biotinylation. We send these
tetramers out with a very high expectation of success. However, the ultimate test
is the binding of the tetramers to the specific T-cell. Unfortunately, we do not
have the resources to perform this test. If you experience problems with your
tetramer, please refer to specific question in the section. If your question is
not answered here please refer to the following section
for contact information.
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Peptide
affinity and tetramer production
One of the factors that determines the success of the tetramer production is the
binding affinity of the peptide for the MHC class I molecule. We do not have specific
numbers that would represent a binding threshold for the peptide, but hope to
have some information regarding this topic in the near future.
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Modified
Termini
No, unless it is otherwise known from explicit binding experiments that the modification
DOES NOT affect binding. Acetylated amino termini and extensions to aid solubility
can cause serious problems during the folding reaction
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How
much tetramer
Typically we ship 200 ug or 4.2nmoles of class I complex conjugated to streptavidin.
Typical working dilution is 1:100; therefore, the amount shipped is sufficient
for approximately 200-400 staining reactions. Requests for large scale amounts
are subject to additional review following initial tetramer studies.
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Average
Shelf-life
We can't answer that question because it varies with each peptide. However, the
tetramers that we have had the most experience with are stable at 4C for greater
than 6 months.
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Monomer
Storage
We store biotinylated MHC/peptide complexes (before addition of streptavidin)
at ‚80C. The monomer is stored in 100 ul aliquots at 2 mg/ml in 1xPBS with 1 ug/ml
Leupeptin, 1 uM Pepstatin and 2 mM EDTA.
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Tetramer
Storage
MHC class I tetramers should be stored at 4C. We have not extensively tested the
effect freezing has on the quality of tetramers prepared with PE or APC. However,
the manufacturer of the Streptavidin-PE and ‚APC cautions against freezing.
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Tetramer
Solution
Tetramer are stored in PBS with leupepin, pepstatin A, EDTA and sodium azide
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Tetramer
Concentration
The concentration of various tetramers is different depending on which fluorescent
label is used. For most tetramers the approximate concentrations are:
| PE |
200ug/425ul |
approximately 10uM |
| APC |
200ug/275ul |
approximately 15uM |
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Background
Staining
- Titrate your reagents.
Check out the effect of
titrating anti-CD8 antibody
- Remove aggregates. Predilute
the tetramer to say a 2X stock in staining buffer and then spin it at high
speed in a microfuge at 4ƒC.
- Add biotin to staining
buffers. If the problem is excess free avidin, you could try blocking by adding
biotin to your staining buffers (100 mM biotin stock in 200 mM Tris base (not
pH adjusted); this will be used at 1:1000).
- Preblock with unlabeled
streptavidin. You could try preblocking with free streptavidin (you could
also try it in your staining buffers)
Finally, you could try using
exclusion. For human cells, put CD14, CD19, etc into one channel, and then gate
on the negative cells. CD4 also fits into this category. Finally, you may have
better results with whole blood staining protocols. You can try staining at 37ƒC.
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Tricolor
CD8-tricolor is readily available, leaving the FITC channel for other reagents
(e.g. CD62L, CD45RA, etc) that may not be available as Tricolor conjugates. For
macaque experiments CD8_PerCP from BD also performs well.
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Negative
Controls
Without compelling justification we do not supply negative controls. Because each
negative control would need a cell line to confirm its functionality, we feel
that this is not an effective use of resources. We highly recommend the use of
negative control animal or patients, either antigen negative or haplotype mismatched.
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Tetramer
Sensitivity
We have observed that working with splenocytes, the background staining can be
as high as 0.5%. It might help to ficoll purify your cells. It also might help
to do adherence depletion. With human peripheral blood, the background levels
can be as low as 0.01%. When working at this level, you need to acquire a lot
of events The better sensitivity is due to lower less background staining.
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Increased
Tetramer Sensitivity
For splenocytes it might help to ficoll purify your cells. It also might help
to do adherence depletion. In addition the use of a third label can increase sensitivity.
Some investigators use the extra channel for exclusion -- either of dead cells
using PI, or non-T cells using a variety of markers. I urge caution, however,
when choosing extra markers, because some of the "cannonical" B and macrophage
markers (e.g. B220 and Mac-1) turn out to be expressed on activated CD8's in the
mouse.
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Tetramer
ELISA
Characterizing the functionality of recombinant T-cell receptors in vitro: a pMHC
tetramer based approach. Tissot et al, Journal of Immunological Methods 236 (2000)
147-165.
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Problematic
Haplotypes
HLA-B*44 and HLA-B*57 do not fold properly, and we have occasional problems
with the biotinylation of H-2Kd allele.
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MHC
Class II Tetramers
We are pleased to announce
the availability of custom-made and premade class II MHC tetramers. Please
click here for more information.
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