Description
Non-specific monocyte binding remains a ubiquitous challenge in flow cytometry. Although the exact mechanism is undefined, literature implicates CD64 interactions with cyanine dyes. However, our data demonstrate that non-specific binding extends beyond cyanine-based fluorophores: certain non-cyanine fluorescent dyes also exhibit this phenomenon. Crucially, we observed selective non-specific staining on monocytes for all tested PE and APC tandem dyes. Human Fc Receptor Blocking Solution is a non-antibody blocking solution and has been formulated to block non-specific binding of PE-Cy7, PE-Cy5, PE-Cy5.5, PE-APC, APC-Cy7, and AF647 which is also a cyanine dye commonly appeared on monocytes and macrophages.This buffer contains specialized human IgG containing 0.05% ProClin 300. It is not recommended to be used for staining human IgG. Handle as biohazard agent.Human peripheral blood leukocytes were either not preincubated (Upper Plots) or were preincubated (Lower Plots) with Human Fc Receptor Blocking Solution as indicated. The cells were then selectively stained with either Mouse IgG1-kappa (PE-Cy5.5), anti-E8L (Ab216584), Mouse IgG1-kappa (PE-Cy7), anti-E8L (Ab216583), Mouse IgG2b-kappa (PE-Cy5), anti-E8L (Ab216565), Mouse IgG2c-kappa (PE-Cy5), anti-HEL (Ab212231) or Mouse IgG2c-kappa (PE-Cy7), anti-HEL (Ab212232). Two-dimensional pseudocolor plots of SSC-A vs Mouse IgG show that the Human Fc Receptor Blocking Solution reduces non-specific binding compared to staining without any blocking reagent. Human peripheral blood leukocytes were either not preincubated any blocking reagent (Top Plots) or were preincubated with competitor Fc Receptor Blocking reagent (Middle Plots) or were preincubated with Human Fc Receptor Blocking Solution (Bottom Plots) as indicated. The cells were then selectively stained with either Mouse IgG (APC-Cy7) (Ab213860), Mouse IgG2b-kappa (PE-Cy5), anti-E8L (Ab216565) or Mouse IgG2c-kappa (PE-Cy5), anti-HEL (Ab212231). Two-dimensional pseudocolor plots of SSC-A vs Mouse IgG show that the Human Fc Receptor Blocking Solution reduces non-specific binding compared to staining without any blocking reagent or with competitor Fc Receptor Blocking reagent.