Nonspecific Antibody Binding via Fcγ Receptors

Symptom

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.

1. 1 Myeloid cells (monocytes, macrophages, dendritic cells, granulocytes) express high levels of Fcγ receptors that bind antibody Fc regions independently of antigen specificity
2. 2 Samples enriched in myeloid cells (bone marrow, peripheral blood, spleen, in vitro differentiation cultures) lack FcR blocking step prior to antibody staining
3. 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. 4 Insufficient incubation time or concentration of FcR blocker fails to saturate Fcγ receptors before adding specific antibodies

1. 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. 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. 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. 4 Optimize FcR blocker concentration and incubation time (typically 5-10 minutes at 4°C) to ensure complete receptor saturation before antibody addition

Related Video (3)

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