Protocol for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids

Keiko Maeda; Katlynn Bugda Gwilt; Stefanie S Schmieder; Nicholas C Zachos; Wayne I Lencer

doi:10.1016/j.xpro.2023.102335

Protocol for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids

STAR Protoc. 2023 May 25;4(2):102335. doi: 10.1016/j.xpro.2023.102335. Online ahead of print.

Authors

Keiko Maeda¹, Katlynn Bugda Gwilt², Stefanie S Schmieder¹, Nicholas C Zachos³, Wayne I Lencer⁴

Affiliations

¹ Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
² Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address: [email protected].
³ Division of Gastroenterology and Hepatology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁴ Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard Digestive Diseases Center, Harvard Medical School, Boston, MA 02115, USA. Electronic address: [email protected].

Abstract

Transcytosis is the primary mechanism by which macro-molecules transverse epithelial cell barriers. Here, we present an assay for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids. We describe steps for establishing human enteroids or Caco-2 cells and plating monolayers. We then provide procedures for a transcytosis and recycling assay and a luciferase assay. The protocol facilitates quantification of membrane trafficking and can be used to probe endosomal compartments unique to polarized epithelia. For complete details on the use and execution of this protocol, please refer to Maeda K et al. (2022).¹.

Keywords: Cell Biology; Molecular Biology.

Grants and funding

P01 AI125181/AI/NIAID NIH HHS/United States