Isotype controls for cytokine staining

Stuart Berzins berzins at
Sun Jun 3 23:39:51 EDT 2007

Dear All,

At the risk of re-igniting the whole topic of isotypes controls, am I
correct in arguing that isotype controls ARE important (or at least helpful)
when examining antigen expression by stimulated vs non-stimulated cells (for
example, cytokine expression by T cells)? That is, providing they are used
alongside all the other antibodies in the normal cocktail to control for
compensation issues.

I understand the limitations of isotypes relating to F/P ratios etc, but
equally, by not using isotype controls, isn't it akin to throwing the baby
out with the bathwater because alternate controls (eg examining expression
on known non-expressing cells) do not adequately control for Fcr-binding and
other 'stickiness' issues that might be unique to the stimulated cell type?

Am I right or wrong? I have searched the archives, but arguments against
isotypes seem to rely heavily on having very similar cells available that
are known not to express the antigen in question (eg for CD34 staining).
That is not the case with activated T cells.

Dr Stuart Berzins
NHMRC RD Wright Fellow
Department of Microbiology and Immunology,
The University of Melbourne,
Parkville 3010,
email: berzins at
Ph (office): +61-3-8344-5706
Ph (lab): +61-3-8344-5704
Fax: +61-3-9347-1540
Mobile: 0427 849 123

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