Voie d'immunisation et séquence d'administration de l ... - TEL

tel-00827710, version 1 - 29 May 2013
4. Notes
1. Although the protocol described here focuses on antigen-specific T cells harvested from human
peripheral blood and from mice, similar procedures can be applied to other non-human samples
[25-27] and to other tissues (e.g., tumor infiltrating lymphocytes, TILs). For human peripheral
blood, prepare PBMCs using Ficoll separation. For tissue-based applications, we recommend
including a CD45 staining in one of the channels in order to segregate CD45-positive
hematopoietic cells, and decrease noise in the assay.
2. Although the protocol described here focuses on HLA A2 individuals as example, it can be applied
to any HLA specificity without modification. Moreover, enrichment protocols would also be
applicable for CD4 + T cell, NK-T and γδT cell populations, using respective multimer reagents.
3. Although the protocol described here is based on the combination of PE-labeled multimers and anti-
PE microbeads, you can similarly stain with APC-labeled multimers and enrich with anti-APC
microbeads.
4. When establishing the assay on human samples, we found a better recovery of rare specific T cells
when using MS columns, regardless of the number of loaded PBMC used (1x10 7 -4x10 8 starting cell
populations tested). Exceptions concern TILs, for which you need to use LS columns in order to
avoid clumps and blockage of the column. Similarly, for mouse experiments, LS columns are
recommended due to potential of stromal tissue from lymph nodes and spleen to clog the columns.
5. This recipe was chosen based on our experience in the lab. Other conventional sorting buffers can
be used, but may result in slightly different background signals. Note that sodium azide should be
omitted if planning to cultivate the cells or perform functional assays.
6. Concerning MHC multimers, there are two options. Commercial vendors exist and will sell off-the-
shelf reagents as well as generate custom materials. Providers include Beckman Coulter,
ProImmune and Immudex. The alternative and recommended option is to prepare your own
monomers, thus facilitating multimerization with streptavidin coupled to your desired fluorescent
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