B cells can express B cell receptors (BCR) with different immunoglobulin heavy chain isotypes, such as IgM, IgD, IgG, IgA, and IgE each of which plays a unique role in the immune response. Understanding these roles is crucial for research into immune system function and diseases. B cells expressing different BCRs can be studied using flow cytometry, a technique for analyzing cell characteristics, widely used in immunology to assess cell populations and functions. Different BCRs can be labeled using fluorophore-conjugated antibodies targeting individual immunoglobulin heavy chain isotypes. In flow cytometry, researchers often use Fc receptor (FcR) blocking agents to prevent non-specific binding of antibodies to Fc receptors on cell surfaces. FcRs are proteins found on various immune cells, including B cells, natural killer cells, macrophages, and dendritic cells. Blocking these receptors ensures that the antibodies used in the assay bind specifically to their intended target rather than to FcRs on the cells. FcR blocking agents often contain human immunoglobulins, and can, therefore, interfere with the accuracy of detecting BCR heavy chain isotypes on B cells. This study evaluates the impact of FcR blocking agents on the detection of BCR heavy chain isotypes (IgM, IgD, IgA1-2, and IgG1-4).
We collected blood samples from four healthy volunteers and isolated peripheral blood mononuclear cells (PBMCs) from these samples. The PBMCs were then treated with five different FcR blocking agents: normal mouse serum, human AB serum, and three commercially available anti-human FcR blocking agents. To assess the impact of washing on detection accuracy, we divided the samples into two groups: one was washed after treatment with the blocking agents, and the other was not.
The use of normal mouse serum did not affect the detection of any BCR isotypes, making it a reliable choice for flow cytometry. However, the three commercially available anti-human FcR blocking agents negatively impacted the detection of IgG1-4+ B cells. Human AB serum interfered with the detection of multiple BCR isotypes, including IgM+, IgA1-2+, and IgG1-4+ B cells. Washing the cells before staining partially restored the detection of IgG isotypes, and washing after treatment with human AB serum completely restored the detection of IgA1-2+ B cells. Despite the benefits of washing, we observed that it led to increased background signals and reduced the overall effectiveness of the FcR blocking agents.
Therefore, our study suggests that normal mouse serum is the most reliable FcR blocking agent for detecting various BCR heavy chain isotypes expressed by B cells. It eliminates the need for washing steps, thereby simplifying the process and maintaining accurate detection. These findings provide practical solutions to the challenges associated with using FcR blocking agents in flow cytometry panels for B cell analysis, enhancing the accuracy and reliability of B cell immunology studies.