Blocking Fc receptors for flow cytometry

While, in theory, using antibodies against Fc Receptors (FcRs) is the best way to eliminate unwanted signals mediated by FcR binding, you may not always have a well-optimized antibody pool against FcRs sitting right in front of you for your experiment. Furthermore, such a blocker may not be applicable when you have one of the FcRs, such as CD16, CD32 or CD64 to measure in your channel. In this blog, we share an "old school" way of blocking by just using a serum that is routinely available in most labs.

The blocking solution was simply prepared by adding healthy human serum to PBS to a final concentration of 10%. To make a comparison with another routinely available material, namely fetal bovine serum (FBS), we also prepared a 10% FBS solution in PBS. PBMCs from a healthy donor were incubated 10 minutes with either of these blocking solutions or PBS as a non-blocking control followed by a 30-minute incubation with antibodies in incubation buffer (0.5% BSA in PBS) and three washes with FACS washing buffer (5mM EDTA in PBS).

For analysis, monocytes and lymphocytes were gated using forward and side scatter. To eliminate doublets, single cells were gated by using forward scatter height and width followed by side scatter height and wide (Fig1 A).

As a result, while an expected high percentage (16.7%) of non-specific CD3^dim CD11b^bright signal mediated by FcRs was detected in the non-blocked sample (Fig.1 B left), blocking with 10% of either FBS or human serum significantly reduced the unwanted signals (Fig.1 B middle and right). To further compare the effects of blocking between FBS and human serum, the CD11b^bright population was gated and analyzed for the CD3 signal under a histogram (Fig.2).  A slightly right shift of the CD3 signal in the sample blocked with FBS indicates that human serum has a better blocking efficacy than FBS.

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