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In high-parameter multicolor flow cytometry, achieving optimal resolution remains a challenge even with rigorous sample preparation and precise compensation. A primary source of data distortion is the intrinsic affinity between antibody Fc regions and cellular Fc gamma receptors (FcγRs, such as CD16, CD32, and CD64). This phenomenon—prevalent in myeloid lineages (monocytes, macrophages, dendritic cells), NK cells, and activated lymphocytes—induces persistent non-specific background staining. These artifacts not only precipitate false-positive results but also obscure the detection of low-abundance antigens, compromising population resolution and experimental clarity. While conventional mitigation strategies, including specialized Fc-blockers, serum pre-treatment, and high-concentration protein buffers, are standard practice, they often fail to achieve exhaustive blockade of FcγR interactions. Furthermore, these exogenous reagents introduce additional technical variables and streamline-inhibiting steps that limit data reproducibility and assay sensitivity.
To address these challenges, Creative Diagnostics has utilized its proprietary recombinant protein engineering platform to develop the CleanFc™ series. By introducing precise molecular modifications to the Fc domain, we have silenced FcR-binding at the source. This "Fc-silent" antibodies ensures the highest degree of data integrity and unmasked signal sensitivity, providing the most authentic representation of your biological samples.
The CleanFc™ series antibodies are precisely redesigned at the genetic level through protein engineering, endowing them with an "immunologically silent" profile.
Utilizing advanced recombinant technology, we have introduced specific amino acid substitutions within the Fc domain. This design completely eliminates physical binding to the FcγRI, FcγRII, and FcγRIII families, while fully preserving the antigen recognition and binding capacity of the Fab domain.Importantly, affinity for FcRn (the neonatal Fc receptor) is retained, ensuring structural integrity and excellent stability of the antibody both in solution and in vivo. Furthermore, nonspecific recognition by complement components such as C1q is avoided, thereby eliminating complement-mediated interference at the source.
Fig. 1 Mutations introduced in the Fc portion of mAbs to render them CleanFcTM
All CleanFc™ antibodies are expressed in standardized systems. This recombinant approach eliminates the inherent heterogeneity and batch-to-batch variability associated with traditional hybridoma production, ensuring absolute experimental reproducibility.
Fig. 2 Binding to human FcgRI by SPR
IgG: Wildtype Rabbit IgG;
LALA/Agly: CleanFcTM Rabbit IgG
Fig. 3 Binding assay of Fc-intact and
CleanFcTM variants of mouse IgG2a and human
IgG1 to FcRs on BMDMs assessed by FC
Fig. 4 Thermal stability of CD20 antibodies measured by differential scanning fluorimetry.
Representative melt curves (normalized first differential) are shown for: (Left) wild-type IgG1, IgG2, IgG3 and IgG4 and (Right)
wild-type IgG1, L234S/L235S/G236R (STR), L234A/L235A (LALA), and N297A (aglycosyl).
Fig. 5 Flow cytometry of BMDMs stained with wild-type (Left) and CleanFc™ (Right) and isotype control antibodies.
Myeloid cells express high levels of Fcγ receptors (notably CD64 and CD32). Conventional antibodies frequently result in substantial background signal and may even lead to false-positive population assignment. The use of CleanFc™ antibodies is strongly recommended to eliminate Fc receptor–mediated interference at the source.
| Cell type | Cell Markers |
| Monocytes | CD14, CD16, CD64, CD11b, HLA-DR |
| Macrophages | CD68, CD11b, F4/80, CD80, CD86, CD163, CD206 |
| Neutrophils | CD11b, CD15, CD66b, Ly6G, CD11c |
| Dendritic Cells (DCs) | CD11c, HLA-DR, CD123,CD80, CD83,CD1c, CD141 |
| Myeloid-Derived Suppressor Cells (MDSCs) | CD11b, CD33, Ly6G/Ly6C, HLA-DR |
Lymphocyte subsets are highly heterogeneous. In high-parameter flow cytometry, conventional antibodies may still introduce signal spread and background noise. Fc-silent antibody formats can significantly reduce fluorescence spillover and improve population resolution.
| Cell type | Cell Markers |
| T cells | CD3, CD25, CD69, HLA-DR |
| Helper T cells (Th) | CD3, CD4, CD62L, CD45RA/RO, CCR7 |
| Cytotoxic T cells (Tc) | CD3, CD8, GranzymeB, Perforin, IFNG |
| Regulatory T cells (Treg) | CD4, CD25, FoxP3, CD127, GITR, CTLA-4 |
| B cells | CD19, CD20, CD22, CD27, IgD, IgM, CD38, CD138 |
| NK cells | CD3, CD56, CD16, NKp46, CD158, CD94 |
Tumor-infiltrating immune cells often exhibit elevated Fc receptor expression and are associated with increased cellular debris and non-specific binding. Fc-silent antibodies enable ultra-low background detection and significantly improve the identification of low-abundance or low-expression markers.
| Cell type | Cell Markers |
| Exhausted T cells | PD-1, Tim-3, Lag-3, TIGIT, CD39 |
| Tumor-associated macrophages | CD206, Arg-1, CD11b, CD163 |
| Activation / Exhaustion balance | CD226, CD28, CD134, CD137 |
| Cell type | Cell Markers |
| Hematopoietic Stem Cells (HSCs) | CD34, CD38, CD90, CD45RA |
| Mesenchymal Stem Cells (MSCs) | CD73, CD90, CD105, CD34, CD45 |
| Endothelial Progenitor Cells (EPCs) | CD34, CD133, VEGFR2 |
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