Isostype Controls in Flow

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coltifer
coltifer's picture
Isostype Controls in Flow

Simple question I think...

Do you need to use an isotype control with the same color fluorochrome as the antibody you used?

For instance, if I have an antibody PE-CD3, does my isotype control for that antibody have to be PE?

marcus muench
marcus muench's picture
Simple answer, yes.

Simple answer, yes.

The purpose of the isotype control is to let you know the degree of background staining a non-specific antibody labeled with the same fluorochrome has on your cells. You need the same fluorochrome for two reasons: 1) Without it, the degree of binding can't be measured in the proper channel, e.g. it doesn't help to know the background staining in the FITC channel for your PE-antibody. 2) Fluorochromes, themselves, are chemicals with more or less propensity to bind cells non-specifically. The whole point of using a similarly labeled isotype control is to determine the degree of staining that should be considered "negative" since what is positive and negative is all relative depending on your settings. On some cells, e.g. macrophages, the difference between no antibody and an isotype control is notable and you need to know that to avoid declaring something as falsely positive.