Protocol for measuring interorganelle contact sites in primary cells using a modified proximity ligation assay

Autor: Hema Saranya Ilamathi, Sara Benhammouda, Laurent Chatel-Chaix, Marc Germain
Jazyk: angličtina
Rok vydání: 2024
Předmět:
Zdroj: STAR Protocols, Vol 5, Iss 1, Pp 102915- (2024)
Druh dokumentu: article
ISSN: 2666-1667
DOI: 10.1016/j.xpro.2024.102915
Popis: Summary: Interorganelle contact sites regulate lipid metabolism, organelle dynamics and positioning, as well as apoptosis and autophagy. Here, we present a proximity ligation assay (PLA) protocol for measuring the association of two organelles in fixed cells. We describe steps for primary cell culture, primary cell transfection, and the assay itself. We then detail procedures for manual and image J-based analysis of PLA foci. This protocol optimizes the use of assay products and improves the identification of PLA foci labeling actual contact sites.For complete details on the use and execution of this protocol, please refer to Ilamathi et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Databáze: Directory of Open Access Journals