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.

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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.

human trustain image

Human TruStain FcX™ treated (filled histograms) or non-treated (open histograms) THP-1 cells stained with anti-human HLA-DR PE antibody (red) or an isotype control (IgG2a PE, blue). Non-treated cells show false-negative HLA-DR staining due to high binding of mouse IgG2a isotype.

If you are using a secondary antibody that might detect the isotype of either mouse or human TruStain, you might seek out an alternative. The serum of the host primary antibody can be used to block Fc receptors instead. For example, if your antibody is of a rat isotype, you could use rat serum to block non-specific binding of immunoglobulins/antibodies to the Fc receptors.

Monocyte Blocking...

Prevents live monocytes from non-specifically binding to fluorophores.

Monocytes and macrophages can non-specifically bind to fluorophore-conjugated antibodies used in cell surface staining of live cells. While there are some publications which propose this phenomenon may occur due to the function of CD64, Fc blocking does not ameliorate this effect. This effect is also not cyanine-specific. Our testing shows that PE/Dazzle™ 594, which is not a cyanine-based acceptor fluorophore, can exhibit non-specific binding to monocytes. On the other hand, cyanine-based fluorophores like Alexa Fluor® 647 and the cyanine-based acceptors of some of the Brilliant Violet™ tandems do not exhibit non-specific binding to monocytes. Our testing has shown this effect appears to be quite selective for any PE, PerCP and APC tandems. (please note that the tandem fluorophores of competitors were not included in our analysis).

 

BioLegend has now formulated an effective blocking reagent, True-Stain Monocyte Blocker™. It is a non-antibody based blocking solution that has been shown to reduce non-specific monocyte binding due to the fluorophore and does not affect the desirable specific staining of monocytes. To learn more about which fluorophores might require True-Stain Monocyte Blocker™, take a look at the chart to the right. To learn more about this reagent, visit our webpage.

Fluorophore conjugates recommended for use with True-Stain Monocyte Blocker™ Fluorophore conjugates that likely do not need True-Stain Monocyte Blocker™
PE/Cy5 Non-tandems: FITC, PE, APC, Pacific Blue™, PerCP, BV421™, BV510™, Alexa Fluor® 488, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 700
PE/Cy7
PE/Dazzle™ 594
APC/Fire™ 750 Brilliant Violet™ tandems: BV570™, BV605™, BV650™, BV711™, BV750™, BV785™
APC/Cy7
PerCP/Cy5.5
PerCP/Cyanine5.5
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Human PBMCs were stained with 5 µl/test of antibody in 100 µl of cells at 1 x 106 cells/ml with (bottom row) or without (top row) True-Stain Monocyte Blocker™.

Isotype Controls...

Represent the non-specific binding present on cells due to non-specific binding of an antibody to the Fc component or conjugated fluorophore of the antibody. 

Even after blocking Fc receptors, it can be helpful to include an isotype control to see how much non-specific binding your cells may exhibit. For instance, certain cyanine dyes also can bind to cells independent of Fc receptors. In these scenarios, isotype controls would also help indicate non-specific binding.

An isotype control matches the constant region of the antibody to the antibody you plan on testing. However, the Fab portion of an isotype control is raised to be low affinity and against a target that is not likely to be found on the cell type being analyzed (i.e., Dinitrophenyl or Keyhole Limpet Hemocyanin). Essentially, if an isotype control binds to your samples, it’s through the Fc region. This helps tell you how much of the stain of your test antibody (which possesses the same constant or Fc region as the isotype control) is due to the Fc region of the antibody being recognized non-specifically.

You should also make sure to use the same amount of test and isotype control antibody. If the antibody is provided in test size format, identify the microgram quantity of the antibody for that volume and obtain an equal mass of the isotype control. Avoid purchasing isotypes and test antibodies from different companies, as the fluorophore:protein (F:P) ratio may not be consistent between different manufacturers.


View all of our isotype controls…

cd11c image

In this plot, a fully stained sample is shown (red) or a control sample where the isotype control was substituted for PE/Cy5 anti-human CD11c (blue) in an FMO. Notice the population in the green box shifts to the right on the x-axis when using an isotype control, indicating some non-specific binding. However, despite this background, the reagents are well-matched to still make distinct gating on the double positive population possible.

Human

Receptor Main ligand Affinity Cell Distribution
FcεRI IgE Very high Mast cells, Basophils, Monocytes, DCs, Neutrophils
FcεRII (CD23) IgE Low B cells, T cells, NK cells, DCs, Eosinophils, Macrophages
FcγRI (CD64) IgG1> IgG2, IgG3, IgG4 High Monocytes, DCs, Macrophages, Neutrophils , Eosinophils
FcγRIIA (CD32) IgG1 and IgG3>IgG2>IgG4 Low-Medium NK cells, DCs, Macrophages, Monocytes, Neutrophils, Platelets
FcγRIIB* IgG4>IgG1, IgG3>IgG2 Low-Medium Monocytes, DCs, Macrophages, B cells, Neutrophils, Basophils, Mast Cells
FcγRIIC IgG1>IgG3>IgG4>IgG2 Low-Medium NK cells
FcγRIIIA (CD16) IgG1>IgG2 and IgG3>IgG4 Low-Medium Macrophages, NK cells, γδ T cells, Monocytes, DCs
FcγRIIIB IgG1 and IgG3>IgG2 and IgG4 Low-Medium Neutrophils, Mast cells, Eosinophils
FcαRI (CD89) IgA1, IgA2 Low Monocytes, Macrophage and DC subsets, NK cells, Neutrophils, Eosinophils
Fcα/μR IgM>IgA High for IgM, medium for IgA Germinal B cells, Follicular DCs.

*Note that FcγRIIB is actually inhibitory and prevents cell activation. To determine if various cell lines express Fc receptors, you may have to check with the company you purchased it from or the literature.

Mouse

Receptor Main ligand Affinity Cell Distribution
FcεRI IgE Very high Mast cells, Basophils, Monocytes, DCs, Neutrophils
FcεRII (CD23) IgE Low B cells, T cells, NK cells, DCs, Eosinophils, Macrophages
FcγRI (CD64) IgG2a> IgG1, IgG2b, IgG3 High Monocytes, DCs, Macrophages, Neutrophils , Eosinophils
FcγRIIB* IgG1 and IgG2b>IgG2a Low Monocytes, Macrophages, B cells
FcγRIII (CD16) IgG2a and IgG2b>IgG1 Medium NK cells, Macrophages, γδ T cells, Monocyte subsets
FcγRIV IgG2a, IgG2b, and IgE Medium Monocytes, DCs, Neutrophils
Fcα/μR IgM>IgA High for IgM, medium for IgA B cells, Monocytes, and Macrophages

References:

  1. Pleass, R. J. 2011. Parasite Immunol. 31:529. Pubmed
  2. Smith, K.G. and Clatworthy, M.R. 2010. Nat. Rev. Immunol. 10:328. Pubmed