Protocol for analyzing transforming growth factor β signaling in dextran-sulfate-sodium-induced colitic mice using flow cytometry and western blotting

Mai Fujiwara; Lucien Garo; Amrendra K Ajay; Alkeiver S Cannon; Panagiota Kolypetri; Shivnarayan Dhuppar; Gopal Murugaiyan

doi:10.1016/j.xpro.2023.102249

Protocol for analyzing transforming growth factor β signaling in dextran-sulfate-sodium-induced colitic mice using flow cytometry and western blotting

STAR Protoc. 2023 Apr 25;4(2):102249. doi: 10.1016/j.xpro.2023.102249. Online ahead of print.

Authors

Mai Fujiwara¹, Lucien Garo², Amrendra K Ajay³, Alkeiver S Cannon¹, Panagiota Kolypetri¹, Shivnarayan Dhuppar¹, Gopal Murugaiyan⁴

Affiliations

¹ Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
² Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA; Boston University School of Medicine, Boston, MA 02118, USA.
³ Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
⁴ Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address: mgopal@rics.bwh.harvard.edu.

Abstract

Transforming growth factor β (TGF-β) is critical to the maintenance of intestinal immune homeostasis. Here, we present techniques for analyzing Smad molecules downstream of TGF-β receptor signaling in dextran-sulfate-sodium-induced colitic mice. We describe colitis induction, cell isolation, and flow cytometric cell sorting of dendritic cells and T cells. We then detail intracellular staining of phosphorylated Smad2/3 and western blotting analysis of Smad7. This protocol can be performed on a limited number of cells from many sources. For complete details on the use and execution of this protocol, please refer to Garo et al.¹.

Keywords: Cell Biology; Cell Isolation; Flow Cytometry/Mass Cytometry; Immunology; Model Organisms; Molecular Biology; Signal Transduction.

Abstract

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