Do I need to perform Fc blocking when using rabbit or mouse antibodies in flow cytometry?

Human Fc-gamma Receptor 1a (FCGR1A) exhibits strong cross-reactivity with rabbit IgG, and mouse IgG2a, IgG2c, and IgG3. Of course, mouse Fc-gamma Receptors will react with mouse antibodies. In flow cytometry experiments using live cells, this can result in unintended signal when the antibody is bound by the FCGR1a protein and is not distinguishable from the binding of the antibody to its intended epitope. To prevent this, Fc blocking can be performed using commercially available reagents to saturate the Fc receptors prior to the addition of your antibodies of interest.

Not all cells express high levels of Fc-gamma receptors, so not all cells require Fc blocking. Cells that express high levels of FCGR1a are typically of myeloid lineage, in both primary cells (e.g., PBMCs) and cell lines (e.g., THP-1, OCI-AML2, U-937, HL-60, etc.). The correct way to check for unintended Fc-gamma receptor binding is to use an isotype control that matches the isotype of the antibody of interest. Any strong signal observed with the isotype control will indicate unintended binding occurring with the Fc domain (or other portions of the antibody).

Please note that Fc blocking does not need to be performed on fixed cells. Once cells are fixed with aldehyde or alcohol, the FCGR1a protein is no longer capable of binding to the rabbit IgG.     

Last updated: September 12, 2024