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

STAR Protocols (Jun 2023)

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

Affiliations

Mai Fujiwara: Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
Lucien Garo: 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
Amrendra K. Ajay: Renal Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
Alkeiver S. Cannon: Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
Panagiota Kolypetri: Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
Shivnarayan Dhuppar: Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
Gopal Murugaiyan: Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA; Corresponding author

Journal volume & issue: Vol. 4, no. 2
p. 102249

Abstract

Read online

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

About the journal

Abstract

Keywords