The isotype must have the same conjugate as the test antibody. Additionally, the protein concentration and fluorophore:protein ratio (if the conjugate is a fluorophore) must match.
Antibody species and class
The isotype and test antibody must be from the same host species and of the same class (i.e. mouse IgG2b), which impacts how it interacts with Fc receptors or other cell components.
The only difference between the isotype and test antibody should be antigen specificty: isotype controls do not specifically bind to any antigens present in cells/samples of interest.
If an isotype control produces high background signal, it may be due to Fc receptor (FcR) binding to the Fc portion of antibodies. Samples containing high frequencies of cell types expressing FcRs (granulocytes, B cells, macrophages, dendritic cells, and others) may require the use of FcR blocking reagents. Utilize Human TruStain FcX™, a blend of human IgG antibodies that use their Fc portions to block FcRs, and Mouse TruStain FcX™ PLUS, an antibody specifically directed against CD16 (FcγRIII) and CD32 (FcRγII) through its antigen-binding domain, to prevent background signal from FcR binding.