Introduction to Fc Receptors (FcR) and Their Impact on Flow Cytometry Results

Antibodies are Y-shaped molecules consisting of Fab and Fc regions. The Fab region is responsible for specific binding to target molecules, while the Fc region interacts with effector molecules or cells.

Figure 1: Structure of an Antibody

Fc receptors (FcR) are crucial receptors in immune regulation, primarily expressed on innate immune cells. They recognize and bind to the Fc region of antibodies, triggering an immune response.

Based on the types of immunoglobulin (Ig) they bind to, FcRs can be categorized into five classes: IgG (FcγR), IgE (FcεR), IgA (FcαR), IgM (FcμR), and IgD (FcδR).

Figure 2: Fc Receptor Classification for IgG Antibodies

The Fc receptor for IgG antibodies is FcγR, which can be further divided into three subfamilies based on their affinity for the Fc fragment: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16).

Among them, FcγRI has the highest affinity for IgG, followed by FcγRIII and FcγRII.

· FcγRII can be further divided into three subclasses: FcγRIIa (CD32a), FcγRIIb (CD32b), and FcγRIIc (CD32c).

· FcγRIII can be further divided into two subclasses: FcγRIIIa (CD16a) and FcγRIIIb (CD16b).

Cells expressing Fc receptors include B lymphocytes, dendritic cells, monocytes, macrophages, NK cells, neutrophils, eosinophils, human platelets, mast cells, and basophils. These cells express multiple types of Fc receptors on their surface, with monocytes and macrophages being particularly notable.

Flow cytometry utilizes fluorescently labeled antibodies to distinguish different cell subsets. The specificity of antibody binding depends on the unique variable regions of the Fab region in each antibody clone. However, the Fc region of antibodies can also bind to Fc receptors on the cell surface, which is referred to as non-specific binding. It is important to note that this non-specific binding signal is not subtracted when using isotype controls, which can lead to increased background or even false-positive signals in flow cytometric analysis.

Figure 3: Impact of FcRs on Detection of Mouse Peritoneal Macrophages

As shown in the figure, mouse peritoneal macrophages were analyzed for the expression of CD3-PE/Cyanine7 and CD11b-FITC, both with and without blocking using Purified Anti-Mouse CD16/32 Antibody.

From the figure, it can be observed that macrophages, when blocked, do not show any expression of CD3, which is consistent with the characteristics of macrophages themselves. In the unblocked samples, most cells show double-positive staining for CD3 and CD11b, which does not align with the inherent characteristics of macrophages. Therefore, Fc receptor blocking is essential for the detection of samples with high Fc receptor expression.

For cells expressing a high number of Fc receptors, we can incorporate Fc receptor blocking agents into the experimental protocol to reduce background caused by non-specific binding of these receptors.

· For human samples, we can use E-AB-F1236A as the blocking agent.

Catalog number: E-AB-F1236A

Product name: Purified Anti-Human CD16 Antibody

· For mouse samples, we can use E-AB-F0997A as the blocking agent.

Catalog number: E-AB-F0997A

Product name: Purified Anti-Mouse CD16/32 Antibody