Flow Cytometry
Selection of Isotype Controls in Flow Cytometry Experiments
Source: Elabscience®Published: Jan 17,2024
Antibodies sometimes bind to certain components within cells due to non-specific protein interactions, as they bind to fats through hydrophobic interactions, and may also bind to some antibody receptors on the cell surface. For example, IgG subtype antibodies bind to Fc receptors on the cell surface, such as CD16, CD32, and CD64. Under ideal conditions, various types of non-specific binding can be blocked by proteins (BSA, milk, or animal serum). The binding of Fc receptors can be blocked by pre incubation with serum containing immunoglobulins. Therefore, the non-specific interactions between antibodies and their binding to Fc receptors can be compared with the same subtype of antibodies that are unrelated to antigens, i.e., Isotype Control. So how to choose an Isotype Control when conducting Flow Cytometry experiments?
Generally, antibodies with the same species, subtypes, and chains as the primary antibody are selected. For example, the FITC labeled human CD56 antibody, which comes from Mouse IgG1, κ Chain, then its Isotype Control should be FITC labeled Mouse IgG1, κ Chain.
If the antibody is in a combination form, such as purified primary antibody combined with fluorescent labeled secondary antibody, then an Isotype Control of the primary antibody should be selected. For example, the purified antibody of CD86 comes from Mouse IgG1, κ Chain, its Isotype Control is purified Mouse IgG1, κ Chain. If the secondary antibody used in the experiment is PE labeled anti mouse IgG1, then the staining method is as follows:
- Sample tube: Purified CD86 + PE labeled anti mouse IgG1 (secondary antibody) + Sample.
- Isotype Control tube: Purified Isotype Control (Mouse IgG1, κ Chain) + PE labeled anti mouse IgG1 (secondary antibody) + Sample.
If a perfectly matched Isotype Control cannot be found, while maintaining the same source, fluorescent labeling, and immunoglobulin category, the priority selection order should be different sub-chains and subtypes. For example, the source of a certain antibody is Mouse IgG2 а,κ Chain, if identical Isotype Controls cannot be found, priority should be given to selecting Mouse IgG2a with the same fluorescent label. If a suitable fluorescent labeled Mouse IgG2a cannot be found, then prioritize choosing Mouse IgG2b with the same fluorescent label. If Mouse IgG2b is still not found, then Mouse IgG2 with the same fluorescent label can be selected as the Isotype Control.