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Artifacts Arising from Using Leukocytic Fc Receptor Blocking Buffer.

S V Zubova1, D S Kabanov2, D A Serov2

  • 1Institute of Fundamental Problems of Biology, Russian Academy of Sciences, Pushchino, Russia. zusvet@rambler.ru.

Bulletin of Experimental Biology and Medicine
|December 2, 2016
PubMed
Summary
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Human TruStain FcX buffer impacts measurements of Toll-like receptor 4 (TLR4) and inflammatory markers like NF-κB p50 and TNF-α. Researchers must consider its effects when using it with isolated cells or whole blood.

Area of Science:

  • Immunology
  • Cell Biology

Background:

  • Fcγ receptor blocking solutions are used to prevent non-specific antibody binding in flow cytometry and other immunoassays.
  • Accurate quantification of cell surface and intracellular markers is crucial for understanding immune responses.

Purpose of the Study:

  • To investigate the effects of Human TruStain FcX buffer on the measurement of Toll-like receptor 4 (TLR4) levels.
  • To assess the impact of the buffer on intracellular staining of NF-κB p50 and TNF-α synthesis.
  • To evaluate the buffer's influence on both isolated human monocytes and whole blood cells.

Main Methods:

  • Flow cytometry was used to measure TLR4 expression.
  • Intracellular immunofluorescent staining was performed for NF-κB p50.
  • Tumor Necrosis Factor-alpha (TNF-α) synthesis was analyzed.
Keywords:
Fcγ receptorsHTA125Human TruStain FcXNF-κB p50TNF-α

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  • Experiments were conducted on human isolated monocytes and whole blood cells.

    • Main Results:

    • Human TruStain FcX buffer demonstrated an influence on the measured parameters, including TLR4 levels, NF-κB p50, and TNF-α synthesis.
    • The extent of this influence varied between isolated cells and whole blood samples.

    Conclusions:

    • The use of Human TruStain FcX buffer can affect the outcomes of immunological assays.
    • Careful consideration of the buffer's impact is necessary when designing experiments involving isolated cells and whole blood.
    • Researchers should validate the appropriateness of Fcγ receptor blocking solutions for their specific experimental conditions.