Isolation of Immune Cells from Adipose Tissue for Flow Cytometry

Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Beige adipocytes Brown adipocytes White adipose tissue Brown adipose tissue Alternatively activated macrophages Eosinophils Group 2 innate lymphoid cells 

References

  1. 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 CrossRefPubMedGoogle Scholar
  2. 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 CrossRefPubMedGoogle Scholar
  3. 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. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 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 CrossRefPubMedGoogle Scholar
  14. 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 CrossRefPubMedGoogle Scholar
  15. 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 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Pathology & ImmunologyWashington University School of MedicineSaint LouisUSA

Personalised recommendations