Artifacts and non-specific staining in flow cytometry, Part I

If you add your antibody, lets say anti-CD3-epsilon antibody, to your cell solution you’d expect that only T cells will be labeled, right? Well, if it were so easy then it wouldn’t be biology!

 

antibodiesIn this first half of the two part blog, I will talk about the two reasons, namely unspecific binding and Fc-receptors, which most people think of when they talk about non-specific binding in flow cytometry.

Some lesser known, but intriguing and important, ‘pseudo-artifacts’ will be covered later in Part II of the blog.

 

(1) Unspecific binding

Unspecific binding is defined as any sticking of an antibody or a fluorochrome to a cell in a fashion that does not require a specifically (currently) defined interaction. This might occur due to electrostatic interactions, glycolipid interaction on the cell membrane, protein-protein interactions and DNA binding.

As such unspecific binding of a cell depends heavily on the surface area (for surface stains) and/or its volume (intracellular staining). For example a cell with twice the size (as seen in the FSC) has 4-times the surface area (SF = 4pi r2) and 8-times the volume (V = 4/3pi r3) and consequently the unspecific binding will be 4 to 8-times higher. So, if you see the whole population shifting a bit in your histogram, you might want to check the scatter of the cells. For example, activated cells start proliferating, which increase their cell size along the way.

 

Aggravating factors and potential solutions:

Antibody amount: A surplus of antibody can increase the non-specific binding, leading to a reduction in the separation of your positive cells and reducing the signal:noise ratio.

Þ Potential solution: Titrate your antibody. As a starting point, antibodies with the same fluorochrome conjugate can often be used at similar concentrations.

Extracellular matrix/cell content: All cells bind proteins including antibodies to some degree via various interactions

Þ Potential solution: Addition of protein to the wash and staining solutions will cover many of these binding sites. Most staining protocols include BSA or serum (either human or FCS) for this purpose.

Dead cells: Dead cells are notorious for non-specifically binding antibodies and appear very ‘sticky’. This is partially due to DNA, but including DNAse would only partially solve the problem.

Þ Potential solution: A live/dead differentiation should be included, if possible, in every staining. Dead cells cannot be entirely separated just by FSC/SSC characteristics, especially not after fixation. Keep in mind though, that fixation of your cells after staining with e.g. PI or 7AAD will partially permeabilize all your cells, so that PI or 7AAD can leak out of the labeled cells to other cells eventually homogenously staining all your cells. In the case of 7AAD this can be avoided by inclusion of non-fluorescent actinomycin D (Schmid et al.). However, nowadays multiple live/dead discriminating reagents are available that can be fixed, thereby stopping potential leakage and avoiding this problem altogether.

 

(2) Binding of antibodies to Fc-receptors:

Obviously Fc-receptors (FcR) bind antibodies with high specificity, but the common misconception is that this is solely species-specific. However, FcRs from one species readily bind antibodies from other species to varying degrees. For example, hamster anti-mouse CD3-epsilon (clone 145.2C11) can bind to all mouse FcRs (Wingender et al.).

Þ Potential solution:

(a)   Fab or F(ab)2 fragments: Utilizing antibodies without their Fc-end avoids the problem altogether, but most commercially available antibodies do contain their Fc part.

(b)   ‘Fc-Block’: Adding antibodies that are specific for particular FcRs that block the undesired interaction with your experimental antibody. However, the ‘Fc-block’ commonly used for mice is the blocking monoclonal antibody 2.4G2 (rat IgG2b kappa) which is specific for mouse Fc-gamma-RII  (CD16) and Fc-gamma-RIII (CD32). Therefore, other FcRs are not directly blocked by 2.4G2. However, the majority of commercial antibodies are of an IgG subtype, most of the potential unspecific Fc-binding will be blocked by 2.4G2. Similar products for staining of human cells are widely available.

(c)     Unconjugated antibody: Adding unconjugated antibody of the same species and isotype as your experimental antibody to your staining cocktail will saturate most potential FcR binding sites.

As a positive side effect, adding unconjugated antibodies, either 2.4G2 or any other isotype, to your stain will incidentally also saturate most other potential unspecific bindings, as they were outlined under (1). Therefore, adding unconjugated antibody to your surface and also your intracellular staining cocktails will reduce unspecific binding.

 

So much for part one. As always, corrections and comments are highly welcomed.

 

References:

Schmid, I. et al., 2001. Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. J Immunol Methods, 247(1-2), pp.175–186.

Wingender, G. et al., 2006. Rapid and preferential distribution of blood-borne alphaCD3epsilonAb to the liver is followed by local stimulation of T cells and natural killer T cells. Immunology, 117(1), pp.117–126.

 





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Gerhard WingenderGerhard Wingender is currently an Instructor at the La Jolla Institute for Allergy and Immunology (La Jolla, CA). His main lab toy is flow cytometry and his research interest involve invariant Natural Killer T (iNKT) cells.

 

 





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References:

Schmid, I. et al., 2001. Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. J Immunol Methods, 247(1-2), pp.175–186.

Wingender, G. et al., 2006. Rapid and preferential distribution of blood-borne alphaCD3epsilonAb to the liver is followed by local stimulation of T cells and natural killer T cells. Immunology, 117(1), pp.117–126.

 

Photo credit: AJC1 / Foter.com / CC BY-NC-SA

About Gerhard Wingender

Gerhard Wingender is currently an Instructor at the La Jolla Institute for Allergy and Immunology (La Jolla, CA). His main lab toy is flow cytometry and his research interest involve invariant Natural Killer T (iNKT) cells.

  • witek

    I am sorry but this is very wrong explanation. First of all, most Fc receptors (with one notable exception in placenta) bind only immunocoplexes and not free uncomplexed antibodies so unspecific staining in flow cytometry is not a problem as long one doesnt mix primary, secondary and cells. Second, dead cells or generally cells that bind antibody unspecifically can be easily distingush by addition of irrelevant fluorescent antibody (e.g. donkey anti-goat 488) during staining. This sounds like a better control than e.g. PI (160 kDa of glycosilated proteins vs. 670 Da fluorescent molecule).