Let's discuss Fc block, which is what most people think of when we talk about "blocking" for flow cytometry.
The issue
Off-target binding of antibody to cells due to conserved features of antibodies.
In addition to binding to the specific target (your marker) via the variable domain, antibodies interact with Fc receptors via the Fc region. Fc receptors are among the most rapidly evolving parts of the genome, and there are extensive differences between mouse and human, for a start. The extent of binding will depend on the species and isotype of the antibody, glycosylation of the Fc region and expression of Fc receptors on your cells. Generally, the strongest interactions (and thus the most off-target binding) will occur with cells expressing the high affinity Fc gamma receptor CD64. This is primarily expressed on macrophages, certain dendritic cell subsets, activated human neutrophils and human monocytes (in mice, CD64 is often used as a marker for macrophages, so cells expressing this usually get defined as macrophages). With mouse, you can also get monomeric binding of IgG to FcγRIV.
Why CD64? CD64 (AKA FcγRI) binds the Fc region of IgG with affinities as low as 10^-8 M (Bruhns Immunobiology 2009). For most other Fc receptors (e.g., CD16, CD32), the affinities are orders of magnitude lower (usually 10^-5 – 10^-6 M). Specific binding via the variable domain, in contrast, usually has affinities between 10^-9 and 10^-12 M. What does that mean? To get a comparable level of binding to CD64, you’d need 10 – 10,000 times as high a concentration of antibody. To get the same binding to low affinity Fc receptors, you’d need 1000 to 10,000,000 times as much. Binding to CD16 and CD32 is essentially a non-issue for monomeric IgG. These receptors a best at binding IgG immune complexes where the aggregation of several IgG molecules onto antigen allows for higher avidity binding.
Why are we using anti-CD16/CD32 for Fc blocking for mouse cells, then? Good question. It doesn’t do much. Compounding the low affinities of the receptors for mouse IgG, we don’t actually use many mouse monoclonals for staining mouse cells. The host species of choice is rat, and most rat antibodies—particularly rat IgG2a—don’t bind well to mouse Fcγ receptors. (There's a nice set of papers on Fc-FcR binding from Friederike Jönsson and Marc Daëron's groups, for instance: Specificity of mouse and human Fcgamma receptors and their polymorphic variants for IgG subclasses of different species - Wang - 2022 - European Journal of Immunology - Wiley Online Library) It’s possible that rat monoclonals targeting mouse CD16/32 are doing as much blocking via their Fc regions as through their variable domains, at least when it comes to the monomeric binding that occurs with flow cytometry.
Antibodies can also bind to cells via other conserved non-Fc regions such as the light chain and specific glycans such as Lewis-X.
The reagents
What is it?
The BD and BioLegend mouse Fc blocking reagents are rat monoclonal antibodies targeting CD16/32 (mouse CD16 and CD32 are identical in the extracellular portion).
It’s not clear what the human reagents are. By the way they interfere with IgG staining, they may simply be specific combinations of human IgG isotypes. Blocking reagents for magnetic bead isolation tend to be serum from the same host species as the antibody coupled to the bead.
Alternatives
For mouse, you can get the 2.4G2 anti-CD16/32 hybridoma from ATCC and produce your own Fc block. This will end up cheaper if you do a lot of flow, and if you don’t purify it, it will contain free light chains that may help block.
Mouse IgG or mouse serum (for blocking mouse cells or for blocking binding of mouse-origin antibodies to human cells)
Rat IgG or rat serum (for blocking binding of rat-origin antibodies)
Normal human serum (for blocking human cells)
Recombinant antibodies that don’t bind to Fc receptors (such as the REA antibodies from Miltenyi)
What it’s supposed to do
Block binding to Fc receptors. More broadly, this is the standard “block” people use, so I think a lot of people expect it to reduce all sorts of non-specific binding.
Examples
Mouse splenocytes:
I had to work pretty hard to get examples of an actual effect with mouse splenocytes and anti-CD16/32 blocking. Note that there is a reduction in non-specific binding to mouse macrophages with FcX, but that mouse serum, which binds all Fc receptors, performs much better.
More typical examples from mouse (minimal effect):
Both Fc blocks reduce fluorescent staining for CD16/32. Blocking of other non-specific binding is unsatisfactory.
I tried to replicate the example BD has on their webpage for 2.4G2 Fc block, where the show that Fc block can reduce non-specific binding of the 53-2.1 clone of anti-CD90.2 to mouse splenocytes. I don't see any non-specific binding with this clone, so there's nothing to block. Admittedly, I'm not using a FITC conjugate, nor am I using a FACScan, and these are C57BL/6 mice rather than BALB/c, so there are some differences. I suspect the biggest difference is that I'm not using a ridiculous amount of antibody (staining here for 1hr at 1:250 dilution).
What about human? With human PBMCs, it's much easier to get non-specific staining. Here's a comparison of different Fc blocking reagents with overnight intracellular staining for CCR7 using about 10x too much antibody.
Note that for this case, the cheap reagents work as well or better.
Does that principle of "more is better" hold true?
Maybe. In this case, yes. With intracellular overnight staining on human PBMCs, there is high potential for non-specific binding, particularly if you use excessive concentrations of antibody (like here). Note what's happening with TCR-alpha/beta--we get brighter staining with the Fc block combination because less of the antibody is being used up binding in a non-specific manner.
Any detrimental effects?
There are a couple of minor issues to watch out for.
1) If you use serum or IgG from the same species to block (e.g., human serum on human cells), you won’t get good staining for IgG. Most likely, you’ll outcompete the binding to your cells by having soluble IgG present. Less commonly, this can also happen with unmatched species if your anti-IgG cross-reacts.
Human CD19+ B cells surface stained for IgG and IgM after pre-blocking (no wash) as indicated. Note the reduction in specific staining for IgM-ve IgG-bright cells and the appearance of a new double positive population with FcX. This is consistent with FcX containing human IgG, which neutralizes some of the anti-IgG, reducing specific staining, while binding to Fc receptors on non-class-switched B cells, producing a false-positive. Since these B cells were in human IgG (serum/blood) previously, they all likely have some IgG bound to Fc receptors on their surfaces, which would account for the slight overall positivity in the absence of FcX. Alternatively, this may be non-specific binding, though the mouse serum block suggests otherwise.
These mouse splenocytes show reduced staining for IgM after blocking with mouse "IgG". This preparation of IgG is likely impure. Note that the IgM-bright B220- population is plasma cells (intracellular IgM staining).
2) Unless specifically prepared as a tissue culture/in vivo grade reagent, Fc block will contain azide as a preservative like other antibodies. If you use it in functional assays or on cells incubated at 37C, you’ll likely get a reduction in viability and cellular responses.
3) FcγR crosslinking has functional effects, both inhibitory and activating. Again, if you’re using Fc block at 37C, this could be an issue.
When do you really need it?
With human blood, you need some sort of Fc block all the time. With mouse cells, if you’ve properly titrated your antibodies, Fc block probably won’t do much on lymphoid tissues. You’ll probably want it if you’re working with tissues where there are more activated macrophages, but mouse serum may work better and end up cheaper.
Bottom line/how to get the best results
The safest option is to pre-incubate your cells with Fc block (or serum) for about 20 minutes before adding your staining mix. Do this for surface staining. However, in many cases, you may achieve similar results by simply adding the block to your staining mix (co-blocking below).
Human PBMCs blocked as indicated with a mix of Fc blocking reagents and stained for 1hr at room temperature (surface staining).
If you’re doing intracellular staining, repeat the blocking again after permeabilization.
Don’t use it on compensation beads. It’ll block binding of the conjugated antibody to the positive beads. See Laura Ferrer-Font’s paper: Panel Optimization for High‐Dimensional Immunophenotyping Assays Using Full‐Spectrum Flow Cytometry - Ferrer‐Font - 2021 - Current Protocols - Wiley Online Library
Reagents used: IgM BUV395
IgM Alexa Fluor 532: prepared in house
Very informative. What would be your thoughts on blocking again after permeabilization, if a pre-blocking step is used prior to surface staining?
Fantastic post, very informational! Quick question about the mouse serum on mouse cells - do you recommend heat-inactivating it?
Great information! Based on the two latest posts in the series looks like mouse serum would be good candidate for replacing both Fc blocking and monocyte blocking reagents.
Excellent material.