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

tel-00827710, version 1 - 29 May 2013
example, CD56 is useful for exclusion of NK cells in human samples when evaluating naive cell
repertoires, but should be used with caution when studying human memory or activated T cell
populations as some cells express CD56 and would thus be lost in the gating strategy. Similarly, it
can be useful to add anti-CD33 and anti-CD34 antibodies when studying T cell populations present
in bone-marrow populations. As indicated above, we deliberately do not included CD4 as we use
the staining of this population as an assessment of background, but this can be added when multiple
free channels are needed for complex multicolor experiments. In the same way, the Dump channel
must to be chosen carefully for mouse experiments: some markers, such as CD11c, may in fact
upregulate on activated T cells.
10. Addition of a viability marker is necessary in order to avoid non-specific background staining on
dead cells. While this may be omitted in some instances in which fresh blood is utilized, it should
be noted that the enrichment columns have an affinity for dead cells. Ideally, select a viability dye
in the same channel as the Dump channel – thus keeping the maximum number of channels free for
phenotypic characterization.
11. At least four fluorescent channels are necessary for careful assessment of enriched T cells: (i) a
Dump channel; (ii) CD3 staining for gating on T cells; (iii) CD8 staining for gating of CD8 + T
cells; and the multimer-conjugated label for the specificity of interest. This can be extended when
differently labeled multimers are included in the same experiment (for reducing non-specific
binding in the assay and/or for multiplexing enrichment). Anti-CD3 can eventually be omitted if
you really need a maximum of free channels for phenotypic characterization. Additional channels
that are available will depend upon the technical specifications of the cytometer and antibodies will
be chosen depending on the experimental questions being evaluated. Note that it is crucial to stain
with multimers prior to washing and staining with other Abs, especially for CD8 and CD4, as some
clones have been shown to influence multimer staining [31].
12. As cells will be fixed to perform intracellular staining, DAPI cannot be used as a viability marker.
We therefore recommend the use of a fixable live/dead cell marker such as Aqua. Note that Aqua
labeling has to be done in azide-free buffer.
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