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

Abstract

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.

Keywords

• Cell Biology
• Cell culture
• Cell-based Assays
• Microscopy
• Molecular/Chemical Probes