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IL-35 Detection in B Cells at the mRNA and Protein Level

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Regulatory B Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2270))

Abstract

Emerging research suggests that IL-35-producing regulatory B cells accumulate in patients and mouse models of pancreatic cancer, one of the most lethal cancers, characterized by late diagnosis, high mortality, and morbidity. Identification of IL-35-producing B cells can be challenging due to the heterodimeric nature of IL-35 and diversity of cell surface markers that define regulatory B-cell subsets across spectrum of diseases. In this chapter, we describe the methods for the isolation of splenic and tumor-infiltrating murine regulatory B cells and subsequent detection of IL-35 by RT-qPCR and intracellular staining, as well as detection of circulating IL-35 by ELISA. We also describe methods for the detection of IL-35-producing human B cells by flow cytometry, RT-qPCR, and immunofluorescence in the context of pancreatic cancer. This chapter should facilitate the study of regulatory IL-35+ B cells in cancer, autoimmunity, and inflammation.

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Acknowledgments

This work was supported in part by R37 CA230786 (Y. P.-G.), University Cancer Research Fund at the University of North Carolina at Chapel Hill, V Scholar Plus Award (Y. P.-G.), Concern Foundation Conquer Cancer Now Award (Y.P.-G), and F31 CA239494-01A1 Ruth L. Kirschstein Predoctoral Individual National Research Service Award (D.M.). The UNC Flow Cytometry Core Facility, the UNC Translational Pathology Laboratory, and the UNC Lineberger Animal Studies Core are supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC LCCC.

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Author notes

  1. Authors Bhalchandra Mirlekar and Daniel Michaud contributed equally to this work.

Authors and Affiliations

  1. Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Bhalchandra Mirlekar & Yuliya Pylayeva-Gupta

  2. Department of Cell Biology and Physiology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Daniel Michaud

  3. Department of Genetics, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Yuliya Pylayeva-Gupta

Authors
  1. Bhalchandra Mirlekar
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  2. Daniel Michaud
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  3. Yuliya Pylayeva-Gupta
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Corresponding author

Correspondence to Yuliya Pylayeva-Gupta .

Editor information

Editors and Affiliations

  1. Department of Medicine, University of Udine, Udine, Italy

    Francesca Mion

  2. Department of Medicine, University of Udine, Udine, Italy

    Silvia Tonon

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Mirlekar, B., Michaud, D., Pylayeva-Gupta, Y. (2021). IL-35 Detection in B Cells at the mRNA and Protein Level. In: Mion, F., Tonon, S. (eds) Regulatory B Cells. Methods in Molecular Biology, vol 2270. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1237-8_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1237-8_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1236-1

  • Online ISBN: 978-1-0716-1237-8

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