Anti-human interleukin(IL)-4 clone 8D4-8 cross-reacts with myosin-9 associated with apoptotic cells and should not be used for flow cytometry applications querying IL-4 expression

Robert Z. Harms, Kiana Borengasser, Vikas Kumar, Nora Sarvetnick

Research output: Contribution to journalArticle

Abstract

Interleukin(IL)-4 is produced by T cells and other leukocytes and is a critical mediator of monocyte and B cell responses. During routine flow cytometry panel validation for the investigation of intracellular cytokines, we observed unique IL-4 expression patterns associated with the widely available monoclonal antibody 8D4-8. Namely, IL-4 (8D4-8) expression was observed in the absence of cellular activation and enhanced following staurosporine exposure. Mass spectrometry analysis of immunoprecipitates from peripheral blood lymphocytes (PBL) revealed that 8D4-8 cross-reacts with the ubiquitous cytoskeletal protein myosin-9. We confirmed these results by western blotting immunoprecipitates, using immunofluorescence among staurosporine-treated Caco-2 cells, and by surface-labeling PBL for 8D4-8 and myosin-9 and analyzing by flow cytometry. Although previously reported from several independent groups, we found no evidence to support the hypothesis that IL-4 is produced by apoptotic cells. Rather, this appears to have been myosin-9. Our data indicate clone 8D4-8 should not be used in the flow cytometric study of IL-4. Furthermore, our work calls for a reevaluation of previous flow cytometric studies that have used this clone for IL-4 analysis and highlights the importance of validation in antibody-based assays.

Original languageEnglish (US)
Article number46
JournalFrontiers in Cell and Developmental Biology
Volume7
Issue numberAPR
DOIs
Publication statusPublished - Jan 1 2019

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Keywords

  • 8D4-8
  • Apoptosis
  • Cross-reacting antibodies
  • Flow cytometry
  • IL-4
  • Interleukin-4
  • Myosin-9

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

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