Selecting the appropriate isotype control may be an important element in flow cytometry experiments. Isotype control antibodies have no specificity for a target cell yet retain all the on-specific characteristics of the antibodies used in an experiment.
The purpose of such a control is to:
- Confirm the specificity of primary antibody binding.
- Rule out non-specific Fc receptor binding or other cellular protein interactions.
ISOTYPE CONTROL USE IN FLOW CYTOMETRY EXPERIMENTS
Isotype control antibodies are optimally developed for cell surface staining protocols and help assess the level of background staining inherent in cell-antibody binding assays.
Two keys to successful isotype control use:
- Due to variations in non-specific binding, the isotype control antibody should ideally match each primary antibody’s host species and isotype.
- When using an isotype control, it is important to use the same antibody concentration (in ug) for both the isotype control and the primary antibody.
Please remember that no isotype control is perfect for every antibody. At eBioscience we attempt to minimize inconsistencies by designing our fluorochrome-conjugated isotype control antibodies within a standard range of fluorophore:protein (F/P ratio) that is similar to our primary antibodies.
Conclusion: Isotype control use is recommended for surface staining.
Isotype controls are a good place to start in optimizing your flow cytometer settings and establishing a data range for the general autofluorescence from a cell labeled with a conjugated antibody. Typically, one would expect an isotype control antibody to show low levels of staining on a particular cell population, (i.e. little shift over autofluorescence), but many times the isotype control does not perform as expected in an intracellular staining experiment, with either extremely low or high levels of non-specific fluorescence.
Because isotype control antibodies are optimized as controls for cell surface staining, there are several potential reasons for inconsistent staining, from inherent differences in the amino acid composition of the two antibodies, to the different amount of fluorophore conjugated to the isotype control versus the experimental antibody (different F/P). Activation of cells may also alter the staining patterns of isotype control antibodies. Hence, we recommend using unstimulated cells or an inherently negative population in a heterogenous cell preparation as more relevant negative controls when staining intracellular targets.
Conclusion: Isotype control use recommended in conjunction with additional negative controls.
What about the fixable viability dyes?
Excluding dead cells from data is recommended for all staining protocols to ensure accurate data. Dead cells can be “sticky”, resulting in non-specific binding, which causes high background staining and false positives. You can find more information in the following post:
If you have some questions about how this technology works, or need any help to set up your assay, our experienced Tech Support Team (all are PhD) will be happy to help you by mail (firstname.lastname@example.org), phone (+34.934464700) or we can visit you. Let’s talk!