Flow Cytometry Controls

 

You may or may not want to utilise isotype controls, but if you do, make sure you have other suitable controls in place, which we shall discuss later. To identify what is particular, isotype controls for surface staining have been created. The proper isotype control is an antibody raised against an irrelevant antigen from the same antibody subclass, using the same conjugated fluorophore, purchased from the same source as your test antibody, and utilised at the same dose as your test antibody. They serve to guarantee that the staining observed is due to specific antibody binding rather than an artefact, to rule out non-specific binding to Fc receptors, and to remove non-specific antibody or fluorophore (such as PE) binding to cellular components. 

Intracellular staining can be more difficult than cell surface staining, and isotype controls may not be acceptable in this situation. In order to correctly calculate your positive population, it is a good idea to have different controls. Unstained samples, negative samples stained with antibody that does not express the antigen of interest, and known positive samples are all examples. Compensation values for multicolor panels must be calculated using single stained compensation controls. Antibody binding beads labelled with your planned multicolor panel can also be used to calculate compensation values. Finally, FMO controls may be used to assess fluorescence spread, gating limits, and prevent decreased sensitivity.