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

tel-00827710, version 1 - 29 May 2013
13. The starting number of cells is a critical point. It is required in order to calculate precursor
frequency; and in most instances it is the determinant of the limit of sensitivity for the assay. For
example, if you suppose your population to be around 10 -6 (meaning 1 cell into 10 6 CD8), you will
need to start from at least 10 7 cells to maximize the possibility of achieving a well-defined
multimer-positive population. Of note, during the enrichment procedure, cell loss is in the range of
10 – 30%.
14. Staining cells with two multimers sharing the same specificity but labeled in different colors
permits a further decrease in the non-specific binders [21]. We and others strongly recommend to
include dual labeling, especially if your aim is to detect ultra-rare populations of cells of variable
avidity. Of course, if you want to pursue with enrichment with anti-PE microbeads, one of the two
colors need to be PE. Otherwise, you can use any fluorochrome combination (up to 25) [21], as
soon as you titrate both streptavidin and multimers before use, and be cautious with settings and
compensations.
15. Concentration of multimers is another important parameter to consider. Optimal concentrations
must be defined for each multimer by titrating on specific cell lines. In general, we recommend
working at high concentrations, i.e. 10-20nM = 3-10µg/mL final concentration (NB: PE Tetramers
~ 500kDa; APC Tetramers ~ 350kDa). Of note, the receptor density of TCR on responding T cells
is also critical. While not thoroughly validated, it may be of interest to evaluate exposure to the
protein tyrosine kinase inhibitor dasatinib prior to staining and/or enrichment as a means of
enhancing tetramer binding [31].
16. For example, if you aim to stain and enrich T cells specific for CMV, Influenza A, and MART1
specific populations at the same time, you will put in the same tube, at the same incubation step, the
following multimers: PE-CMV, PE-Cy7-CMV, PE-Flu, APC-Flu, PE-MART1, APC-Cy7-MART1
(see Note 22 and Figure 6A).
17. Temperature is a critical parameter in multimer staining. It is advisable to assess the effects of
temperature (and time) for each individual system. In our assays, MHC I staining is performed at 4°
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