Don’t be Fooled by Monocyte Non-Specific Antibody Binding

Monocytes and macrophages can exhibit non-specific binding of antibodies and fluorophores used in cell surface staining of live cells. 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. Now you can immuno-label monocytes with confidence, fully trusting your true staining results.

Download the PDF for our scientific poster on True-Stain Monocyte Blocker™.

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There are many fluorophore properties and assay factors that influence the existence or severity of the non-specific binding:

  • PBMCs vs. whole blood vs. pre-lysed blood
  • PMA stimulated vs unstimulated cells
  • Donor-dependent variation
  • Live vs. fixed cells

Though the specific mechanism of the non-specific binding isn’t known, binding of the cyanine acceptor fluorophores to CD64 has been implicated in the literature. However, in our testing, we have demonstrated that the non-specific binding is not a cyanine fluorophore phenomena alone.  PE/Dazzle™ 594, which is not a cyanine-based acceptor fluorophore, also can exhibit non-specific binding to monocytes.  Thus the effect appears to be quite selective for any PE, PerCP and APC tandems we have tested.  The tandem fluorophores of competitors were not included in our analysis.  In our testing, 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. To learn more about which fluorophores might require True-Stain Monocyte Blocker™, take a look at the chart to the right.

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

True-Stain Monocyte Blocker™ prevents monocytes from non-specifically binding to fluorophores

 

 

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™.


Blocking Fc Receptors does not prevent non-specific binding of monocytes

The use of the Fc blocker looked identical to samples stained with no blockers at all. If the non-specific binding were mediated by Fc binding, the Fc blocker (Human TruStain FcX™) alone would have fixed the problem. In instances where only the antibody exhibiting non-specific binding (for example, CD3 APC/Cy7, CD56 PE/Cy5 and CD19 PE/Dazzle™ 594) was used to costain cells with CD14 BV421™, the True-Stain Monocyte Blocker™ alone was sufficient to block the non-specific binding. This can be seen in the data above with True-Stain Monocyte Blocker™.

 

 

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) Human TruStain FcX™ blocking solution.


Multicolor panel components influence the non-specific binding of tandem dyes

Including many of these tandems in a multicolor panel, as is common for panels starting at 6 colors or more, has a variable effect on the extent of the non-specific binding, as if the presence of many of these tandems competes for the non-specific binding sites and can often diminishes the apparent level of non-specific binding of any single reagent. This effect was seen consistently whether using LWB (lysed whole blood), pre-lysed blood or PBMC.

 

Human PBMCs were stained with 5 µl/test of antibody in 100 µl of cells at 1 x 106 cells/ml in a 2-color panel as indicated (top row) or in a 7-color panel (bottom row) as listed in the table below.

Description Clone
CD14 BV421™ HCD14
CD3 APC/Cy7 UCHT1
CD56 PE/Cy5 5.1H11
CD19 PE/Dazzle™ 594 HIB19
CD64 PE/Cy7 10.1
CD4 PerCP/Cy5.5 RPA-T4
HLA-DR Alexa Fluor® 647 L243

True-Stain Monocyte Blocker™ is effective for blocking non-specific binding to murine cells.

 

Mouse resident peritoneal cells were stained with MOPC-21 PE/Cy7 (panels A and B). Monocytes/macrophages show binding to PE/Cy7 as shown in plot A (identified by red arrows). This staining is eliminated when using blocking reagent as seen panel B.

Stimulation diminishes non-specific binding of tandem dyes

In Fig. 2, human PBMC were split into two conditions, unstimulated (Fig. 2A-F) or a 6 hr stimulation with the Cell Activation Cocktail of PMA/Ionomycin with Brefeldin (Fig.2G-I). After stimulation, both conditions were stained with the antibodies listed in Table 2 with appropriate fixation and permeabilization for the detection of intracellular cytokines. Staining cell surface markers in the presence of the True-Stain Monocyte Blocker™ effectively blocked any non-specific binding due to cyanine-based fluorophore tandems. 

The acceptor molecule of PE/Dazzle 594™ is not cyanine-based and exhibited no non-specific binding under either of these sample preparation conditions, whether the cells were stimulated or not. This fluorophore can exhibit some non-specific binding when used only in a 2-color panel, but does not exhibit non-specific binding when used to stain PBMC, LWB or pre-lysed blood in a multicolor panel with other tandem dyes. On the other hand, APC/Fire™ 750, APC/Cy7 and PE/Cy7, all of which are derived from the cyanine family of fluorophores, have variable non-specific binding in multicolor panels on different donors and with different sample prep conditions.

Description Clone
CD8 BV510™ SK1
CD4 BV650™ RPA-T4
CD19 PE/Dazzle™ 594 HIB19
CD3 APC/Fire™ 750 UCHT1
CD14 BV421™ HCD14
CD56 PE/Cy7 5.1H11
IFNγ FITC 4S.B3
TNFα PE MAB11
IL-4 Alexa Fluor® 647 MP4-25D2
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Short term incubation with True-Stain Monocyte Blocker™ does not induce cytokine production

Non-specific binding is only a staining artifact when tandem-labeled antibodies are applied to live cells. Our data indicates that short term exposure (30 minutes) of the True-Stain Monocyte Blocker™ itself does not have any stimulatory effect sufficient to induce cytokine production in PBMCs. The data below demonstrates the lack of IL-4, IFN-γ, and TNFα production under these conditions. In addition, no substantial effect on cell viability or cell activation could be detected in PMA/Ionomycin activated or unstimulated CD4+, CD8+ or CD14+ cell populations (data not shown). 

However, longer incubation times or alternative stimulation conditions, such as TLR or other signaling pathways, would need to be further defined in order to fully assess potential unwanted stimulatory effects from the Blocker solution.

 

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