Nonspecific Antibody Binding via Fcγ Receptors (Flow Cytometry (Sample Considerations))

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Elevated false-positive staining observed in myeloid-enriched samples (bone marrow, blood, spleen, in vitro myeloid differentiation cultures) due to antibody Fc region binding to Fcγ receptors on monocytes, macrophages, dendritic cells, and granulocytes. Common causes: 1) Myeloid cells (monocytes, macrophages, dendritic cells, granulocytes) express high levels of Fcγ receptors that bind antibody Fc regions independently of antigen specificity 2) Samples enriched in myeloid cells (bone marrow, peripheral blood, spleen, in vitro differentiation cultures) lack FcR blocking step prior to antibody staining 3) Some tandem dyes (PE/Dazzle 594, APC/Fire 750, PE/Cyanine7, PE/Cyanine5, PerCP/Cyanine5.5, especially APC/Cyanine7) bind monocytes/macrophages independently of Fc receptors 4) Insufficient incubation time or concentration of FcR blocker fails to saturate Fcγ receptors before adding specific antibodies Solutions: 1) Include FcR blocker (anti-CD16/CD32 for mouse, human Fc receptor binding inhibitor) in staining protocol for myeloid-enriched samples before adding specific antibodies 2) Use True-Stain Monocyte Blocker for samples containing monocytes and macrophages to minimize non-Fc-mediated binding of tandem dyes (PE/Dazzle 594, APC/Fire 750, APC/Cyanine7) 3) Avoid or minimize use of APC/Cyanine7, PE/Cyanine7, PE/Cyanine5, PerCP/Cyanine5.5 tandem dyes on monocyte/macrophage-rich samples; substitute with non-tandem alternatives 4) Optimize FcR blocker concentration and incubation time (typically 5-10 minutes at 4°C) to ensure complete receptor saturation before antibody addition