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When designing your controls, you need to be clear about what service you
expect them to perform. If all your stains will yield a clear separation
between negative and positive populations and you are only measuring
percent positive, then the isotype control is almost academic and preparing
a cocktail with the maximum amount of isotype/fluorochrome used in your
panel will do the job.
However, if you want to use your controls to guide consistent cursor
settings for dim or poorly separated populations, or to determine a
fluorescence threshold for quantitating specific binding, you will want to
be careful to match antibody amounts, isotype, and fluorochrome to the
specific panel members you are controlling, in smaller cocktails that do
NOTcombine two antibodies with the same isotype nor two antibodies with the
same fluorochrome.
In working with cynomolgus monkeys, I would avoid murine IgG2a antibodies
if possible or limit their use to very clearly defined populations, as I
have experienced very high NSB (non-specific binding) of this isotype in a
number of individuals.
Hope this helps.
Karen Henell
Histocompatibility Technologist
Laboratory of Immunogenetics and Transplantation
Oregon Health Sciences University
Portland, OR 97201 USA
henellk@ohsu.edu
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