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Isolation of Immune Cells from Adipose Tissue for Flow Cytometry

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Thermogenic Fat

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1566))

Abstract

Beige and brown adipocytes are thermogenic cells essential for maintaining metabolic homeostasis within white adipose tissue (WAT) and brown adipose tissue (BAT), respectively. Emerging studies indicate that various immune cell types such as alternatively activated macrophages (AAMacs), eosinophils, and group 2 innate lymphoid cells (ILC2s) critically regulate beige and/or brown adipocyte development and activation to protect against obesity and maintain core body temperature. These findings suggest that studies of beige and brown adipose tissue may benefit from traditional immunologic approaches such as flow cytometry of immune cells residing within WAT and BAT. The purpose of this article is to describe an efficient method to isolate immune cells from numerous adipose tissue samples in parallel. The composition, phenotype, and activation state of cells isolated with this protocol may then be assessed by multiple methods including but not limited to flow cytometry. As an example, this article briefly describes a method to identify AAMacs, eosinophils, and ILC2s in adipose tissues.

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References

  1. Cannon B, Nedergaard J (2004) Brown adipose tissue: Function and physiological significance. Physiol Rev 84(1):277–359. doi:10.1152/physrev.00015.2003

    Article  CAS  PubMed  Google Scholar 

  2. Harms M, Seale P (2013) Brown and beige fat: development, function and therapeutic potential. Nat Med 19(10):1252–1263. doi:10.1038/nm.3361

    Article  CAS  PubMed  Google Scholar 

  3. Pfeifer A, Hoffmann LS (2014) Brown, beige, and white: the new color code of fat and its pharmacological implications. Annu Rev Pharmacol Toxicol. doi:10.1146/annurev-pharmtox-010814-124346

    Google Scholar 

  4. Wu J, Cohen P, Spiegelman BM (2013) Adaptive thermogenesis in adipocytes: is beige the new brown? Genes Dev 27(3):234–250. doi:10.1101/gad.211649.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cohen P, Levy JD, Zhang Y, Frontini A, Kolodin DP, Svensson KJ, Lo JC, Zeng X, Ye L, Khandekar MJ, Wu J, Gunawardana SC, Banks AS, Camporez JP, Jurczak MJ, Kajimura S, Piston DW, Mathis D, Cinti S, Shulman GI, Seale P, Spiegelman BM (2014) Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch. Cell 156(1–2):304–316. doi:10.1016/j.cell.2013.12.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wu J, Bostrom P, Sparks LM, Ye L, Choi JH, Giang AH, Khandekar M, Virtanen KA, Nuutila P, Schaart G, Huang K, Tu H, van Marken Lichtenbelt WD, Hoeks J, Enerback S, Schrauwen P, Spiegelman BM (2012) Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 150(2):366–376. doi:10.1016/j.cell.2012.05.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Brestoff JR, Artis D (2015) Immune regulation of metabolic homeostasis in health and disease. Cell 161(1):146–160. doi:10.1016/j.cell.2015.02.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. DiSpirito JR, Mathis D (2015) Immunological contributions to adipose tissue homeostasis. Semin Immunol 27(5):315–321. doi:10.1016/j.smim.2015.10.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nguyen KD, Qiu Y, Cui X, Goh YP, Mwangi J, David T, Mukundan L, Brombacher F, Locksley RM, Chawla A (2011) Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis. Nature 480(7375):104–108. doi:10.1038/nature10653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Qiu Y, Nguyen KD, Odegaard JI, Cui X, Tian X, Locksley RM, Palmiter RD, Chawla A (2014) Eosinophils and type 2 cytokine signaling in macrophages orchestrate development of functional beige fat. Cell 157(6):1292–1308. doi:10.1016/j.cell.2014.03.066

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wu D, Molofsky AB, Liang HE, Ricardo-Gonzalez RR, Jouihan HA, Bando JK, Chawla A, Locksley RM (2011) Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science 332(6026):243–247. doi:10.1126/science.1201475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Molofsky AB, Nussbaum JC, Liang HE, Van Dyken SJ, Cheng LE, Mohapatra A, Chawla A, Locksley RM (2013) Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J Exp Med 210(3):535–549. doi:10.1084/jem.20121964

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Brestoff JR, Kim BS, Saenz SA, Stine RR, Monticelli LA, Sonnenberg GF, Thome JJ, Farber DL, Lutfy K, Seale P, Artis D (2015) Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 519:242–246. doi:10.1038/nature14115

    Article  CAS  PubMed  Google Scholar 

  14. Lee MW, Odegaard JI, Mukundan L, Qiu Y, Molofsky AB, Nussbaum JC, Yun K, Locksley RM, Chawla A (2015) Activated type 2 innate lymphoid cells regulate beige fat biogenesis. Cell 160(1–2):74–87. doi:10.1016/j.cell.2014.12.011

    Article  CAS  PubMed  Google Scholar 

  15. Hams E, Locksley RM, McKenzie AN, Fallon PG (2013) Cutting edge: IL-25 elicits innate lymphoid type 2 and type II NKT cells that regulate obesity in mice. J Immunol 191(11):5349–5353. doi:10.4049/jimmunol.1301176

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by NIH grant AI112023.

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Correspondence to Jonathan R. Brestoff .

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Brestoff, J.R. (2017). Isolation of Immune Cells from Adipose Tissue for Flow Cytometry. In: Wu, J. (eds) Thermogenic Fat. Methods in Molecular Biology, vol 1566. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6820-6_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6820-6_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6819-0

  • Online ISBN: 978-1-4939-6820-6

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