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

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.

SUBMITTER: Ilamathi HS

PROVIDER: S-EPMC10901140 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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

Ilamathi Hema Saranya HS Benhammouda Sara S Chatel-Chaix Laurent L Germain Marc M

STAR protocols 20240222 1

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 identificati ...[more]

PMID: 38393951

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