Ignoring instrument-derived background signal (generally called noise), the cause of background fluorescence can be categorized into three groups: (I) autofluorescence, (II) spectral overlap, and (III) undesirable antibody binding. The level of background in each of these groups depends on, and may be minimized by, several components of the measurement (e.g., antigen of interest, choice of antibody, choice of fluorochrome, cell labeling protocol, and optical configuration of the flow cytometer). With a thorough understanding of how each component may contribute to the background level, sets of control samples can be designed to reliably tell truly positive from negative and other detected signals (i.e., events) in a flow cytometric data set.
|Antigen||A substance that induces the production of antibodies.|
|Autofluorescence||Self-fluorescence; inherent fluorescence of an object.|
|Background||Extraneous signals that can be confused with the phenomenon to be measured.|
|Bleed-through (emission)||Spillover; part of an emission spectrum that overlaps with the peak intensity of another spectrum.|
|Crossreactivity||Binding to an identical or similar epitope than that the antibody was generated against and present on different antigens.|
|Epitope||Portion of the antigen that an antibody is generated against.|
|Event||(Light-derived) electronic signal processed by a flow cytometer and added to the dataset (listmode file).|
|FMO control||Fluorescence-minus-one; a control that includes all antibodies involved in the experiment, except one.|
|Internal negative control||Population of cells that does not express the antigen of interest in a sample that also contains a population of cells that does.|
|Isoclonic control||Mixture of a fluorochrome-conjugated and excess of an identical, but unconjugated antibody.|
|Isotype control||Antibody of the same class (isotype) of immunoglobulin as the specific antibody, but generated against an antigen that is not present on or in the cells under study.|
|Negative cells||Cells that express antigen levels at or below the detection limits of the measurement technology.|
|Nonspecific antibody binding||Binding of an antibody to something that it was not generated against.|
|Relative fluorescence intensity||Amount of fluorescence relative to the instrument settings of the flow cytometer.|
|Resolution index||Degree of separation between a positive and negative population, described as follows:|
|Specific antibody binding||Binding of an antibody to the epitope it was generated against.|
|Spectral compensation||Mathematical method to correct for bleed-through emission.|
|Spectral overlap||Spillover between two (or more) fluorescence emission spectra.|
|Spreading error||Error due to imprecise measurements and spectral compensation.|
|Staining index||Describes how much a positive population is separated from the negative population, as follows:|
|Undesirable antibody binding||User-defined term, but in general: Any type of bond between an antibody and a cell that obscures correct interpretation of the data.|
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.
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.
• 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. Therefore, adding unconjugated antibody to your surface and also your intracellular staining cocktails will reduce unspecific binding.
The fact that Fc-receptors (FcR) bind antibodies is obvious, but lesser known is the fact that some of the fluorochrome linked to your antibody can also bind some FcRs.
It has been reported that R-phycoerythrin (PE) can bind to mouse Fc-gamma-RII (CD16) and Fc-gamma-RIII (CD32) (Takizawa et al.). Furthermore, FcR binding of fluorochromes apparently applies to most or maybe even all cyanine fluorochromes, either alone or in tandem conjugates (Shapiro). So far I found reports for Cy5 (Jahrsdorfer et al.), PE-Cy5 (van Vugt et al.; Steward and Steward; Jahrsdorfer et al.) and APC-Cy7 (Beavis et al.). In this case, human CD64 (Fc-gamma-RI) was suggested to be the culprit of some of the binding (van Vugt et al.; Jahrsdorfer et al.), but binding also to CD64neg leukemia cells has been reported (Steward and Steward), so the role of FcR is not solved for all cases yet.
Þ Potential solution:
(a) PE: For the binding of PE to mouse CD16/32 the use of a 'Fc-block', i.e. adding blocking monoclonal antibody 2.4G2 (rat IgG2b kappa), will avoid the problem (Takizawa et al.).
(b) Cyanine: If you work with FcR+ cells, especially monocytes, you might consider avoiding cyanine-containing fluorochromes for the staining of your cells of interest.
Phycoerythrin (PE) and allophycocyanin (APC) are large proteins of 240kD and 110kD respectively that were original derived from cyanobacteria or red algae. As it turns out these phycobiliproteins are also a specific antigen for some T and B cells. Approximately 0.1% of all mouse B cells recognize PE as antigen in a BCR-dependent manner (Pape et al.; Wu et al.). Similar, about 0.02% of all mouse B cells are APC antigen-specific (Pape et al.). Furthermore, about 0.02-0.4% of all gamma-delta-T cells (mouse and human) recognized PE as a specific antigen (Zeng et al.) as well.
Þ Potential solution: Given their low frequency, these cells only pose a problem if you study tiny subsets of B and gamma-delta-T cells. In that case, you should avoid the use of PE for your cells of interest.
(a) Cross-reactivity of the antibody: Epitopes might be shared between different proteins, i.e. your antibody might not only recognize your protein in question, but recognizes also a similar epitope of another protein. This is for obvious reasons more likely with polyclonal antibodies.
Þ Potential solution: Usage of monoclonal antibodies reduces the risk of such cross-reactivity. If you suspect a cross-reactivity of your antibody, using a different clone for the same epitope will likely solve this problem.
(b) Intracellular biotin: Biotin is an important component of the cell metabolism. Therefore, biotin is present in the cells and the use of a streptavidin for intracellular staining will lead to binding of the streptavidin also to the cellular biotin.
Þ Potential solution: If you need to use a biotin-conjugated antibody for your intracellular staining you could cover all intracellular biotin by incubation of your cells with unconjugated streptavidin (followed by thorough washing) before the addition of your biotin-conjugated antibody.
(c) FITC charge: FITC is a charged molecule and antibodies with many FITC molecules (i.e. high F/P ratio) result in a highly charged antibody that binds, presumably through electrostatic interactions, nonspecifically to cytoplasmic elements (Hulspas et al.). This seems to be mainly a problem with intracellular staining and not with surface stains.
Þ Potential solution: For this reason FITC is not ideal for intracellular staining and you might try your antibody conjugated to a different fluorochrome.
(d) CD205: CD205 (DEC205) is a C-type lectin that is highly expressed on dendritic cells. Recently, it has been demonstrated that PE-Cy5.5 binds with high specificity to mouse CD205 (Park et al.). No staining was observed towards human CD205 and the binding of other Cy5.5 conjugates (PerCP-Cy5.5, APC-Cy5.5 and Cy5.5) to mouse CD205 was much weaker than that of PE-Cy5.5 (Park et al.).
Þ Potential solution: Given the high specificity of the interaction, you should avoid the use of PE-Cy5.5, and to a lesser extent other Cy5.5 containing fluorochromes, when your cells of interest expresses mouse CD205.
Finally, another odd-ball has been reported for APC tandems. Apparently, living cells have some way, which depends on their metabolism, to degrade the APC-Cy7 and APC-H7 tandems, leaving you with an APC signal (Le Roy et al.). APC-Cy7 seemed to be more affected than APC-H7 and monocytes are more active at degrading this signal than lymphocytes.
Þ Potential solution: Given that this requires live cells, fixation of your cell solution after staining will solve this problem. Alternatively, as this degradation requires metabolically active cells, storing your cell solution at 4°C or on ice or adding sodium azide (NaN3) to your storing buffer will reduce the effect.
Ruud Hulspas，et.al., Considerations for the Control of Background Fluorescence in Clinical Flow Cytometry, Cytometry Part B (Clinical Cytometry) 76B:355–364 (2009)
Gerhard Wingender, Artifacts and non-specific staining in flow cytometry, 2013