Protocol for measuring interorganelle contact sites in primary cells using a modified proximity ligation assay
Hema Saranya Ilamathi1, Sara Benhammouda1, Laurent Chatel-Chaix2
1Groupe de Recherche en Signalisation Cellulaire and Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, QC G8Z 4M3, Canada; Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois, Montréal, QC H3C 3P8, Canada; Réseau Intersectoriel de Recherche en Santé de l'Université du Québec (RISUQ), Québec, QC G1K 9H7, Canada.
Abstract:
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.
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