Isotype Controls...

Represent the non-specific binding present on cells due to non-specific binding of an antibody to the Fc component or conjugated fluorophore of the antibody. 

Even after blocking Fc receptors, it can be helpful to include an isotype control to see how much non-specific binding your cells may exhibit. For instance, certain cyanine dyes also can bind to cells independent of Fc receptors. In these scenarios, isotype controls would also help indicate non-specific binding.

An isotype control matches the constant region of the antibody to the antibody you plan on testing. However, the Fab portion of an isotype control is raised to be low affinity and against a target that is not likely to be found on the cell type being analyzed (i.e., Dinitrophenyl or Keyhole Limpet Hemocyanin). Essentially, if an isotype control binds to your samples, it’s through the Fc region. This helps tell you how much of the stain of your test antibody (which possesses the same constant or Fc region as the isotype control) is due to the Fc region of the antibody being recognized non-specifically.

You should also make sure to use the same amount of test and isotype control antibody. If the antibody is provided in test size format, identify the microgram quantity of the antibody for that volume and obtain an equal mass of the isotype control. Avoid purchasing isotypes and test antibodies from different companies, as the fluorophore:protein (F:P) ratio may not be consistent between different manufacturers.

View all of our isotype controls…

cd11c image

In this plot, a fully stained sample is shown (red) or a control sample where the isotype control was substituted for PE/Cy5 anti-human CD11c (blue) in an FMO. Notice the population in the green box shifts to the right on the x-axis when using an isotype control, indicating some non-specific binding. However, despite this background, the reagents are well-matched to still make distinct gating on the double positive population possible.