BioLegend Flow Cytometry Controls

As important as good data and results are, having the right controls to interpret and troubleshoot your experiment is just as critical. Whether you need to gate your populations accurately or identify whether your result is unique or artifactual, controls provide context and clarity.

Fc Blocking Controls...

Block the non-specific detection of the Fc component of all antibodies. It is most appropriate for samples where the cells express Fc receptors that can exhibit non-specific binding of antibody.

Antibodies consist of two heavy chains (blue) and two light chains (red) connected by disulfide bonds. The antigen binding region consists of highly variable regions that allow antibodies to recognize one target out of approximately ten billion (based on recombination events). Beyond these regions, the sequences of antibodies tend to remain relatively constant. Experiments in the 1960s utilized enzymes like papain to help understand the structure of antibodies. Papain is a thiol-endopeptidase that cleaves peptide bonds within the hinge region, creating three fragments: two were found to be identical and named Fab for their ability to bind antigen; the remaining fragment was named Fc for its tendency to crystallize during cold storage.

An antibody has two main functions: to bind foreign antigens and to mediate effector functions of other immune pathways. The latter can be accomplished with the help of Fc receptors, which are present on many cell types, including granulocytes, B cells, macrophages, and dendritic cells. As these Fc receptors recognize the Fc portion of an antibody (and potentially any pathogens attached to the Fab region of the attached antibody), this mechanism triggers several downstream effects, often including phagocytosis, activation or antibody-dependent cell-mediated cytotoxicity. This is commonly associated with Fcγ receptors, which recognize IgG antibodies. Fcε receptors can help launch allergic responses and degranulation.

As these receptors have a propensity to bind the Fc portion of antibodies, they can present false positives in your analysis. To prevent this background staining, we recommend using Human TruStain FcX™ or Mouse TruStain FcX™ PLUS.

Mouse TruStain FcX™ PLUS is an antibody (clone S17011E) specifically directed against CD16 and CD32 (FcγRIII and FcRγII respectively) via the Fab portion of the antibody, with improved Fc blocking capabilities compared to the original mouse TruStain fcX™ (clone 93). Simultaneous detection of CD16 and CD32 with mouse TruStain FcX™ will depend on the clones used and whether the same epitopes are recognized by these reagents. Clone S17011E blocks both clone 93 and 2.4G2, which are also raised against mouse CD16/32.

Human TruStain FcX™, on the other hand, is a proprietary blend of specialized human IgG immunoglobulins that bind to Fc receptors via the Fc portion of the immunoglobulin. Despite occupying these Fc receptors, Human TruStain FcX™ is still compatible with flow cytometric staining with anti-Fc receptor antibodies, such as anti-human CD16 (clone 3G8), CD32 (clone FUN-2), and CD64 (clone 10.1) antibodies.