Protocol to measure centrosome cohesion deficits mediated by pathogenic LRRK2 in cultured cells using confocal microscopy

Elena Fdez; Rachel Fasiczka; Antonio Jesús Lara Ordóñez; Belén Fernández; Yahaira Naaldijk; Sabine Hilfiker

doi:10.1016/j.xpro.2022.102024

Protocol to measure centrosome cohesion deficits mediated by pathogenic LRRK2 in cultured cells using confocal microscopy

STAR Protoc. 2023 Mar 17;4(1):102024. doi: 10.1016/j.xpro.2022.102024. Epub 2023 Jan 13.

Authors

Elena Fdez¹, Rachel Fasiczka², Antonio Jesús Lara Ordóñez³, Belén Fernández³, Yahaira Naaldijk², Sabine Hilfiker⁴

Affiliations

¹ Institute of Parasitology and Biomedicine López-Neyra, Consejo Superior de Investigaciones Científicas (CSIC), 18016 Granada, Spain. Electronic address: [email protected].
² Department of Anesthesiology and Department of Pharmacology, Physiology and Neuroscience, Rutgers New Jersey Medical School, Newark, NJ 07103, USA.
³ Institute of Parasitology and Biomedicine López-Neyra, Consejo Superior de Investigaciones Científicas (CSIC), 18016 Granada, Spain.
⁴ Department of Anesthesiology and Department of Pharmacology, Physiology and Neuroscience, Rutgers New Jersey Medical School, Newark, NJ 07103, USA. Electronic address: [email protected].

Abstract

The present protocol allows for quantification of inter-centrosome distances in G2 phase cells by confocal fluorescence microscopy to determine centrosome cohesion deficits. We describe transfection and immunofluorescence approaches followed by image acquisition and analysis of inter-centrosome distances. This protocol is for adherent A549 cells transiently overexpressing pathogenic LRRK2 and for immortalized murine embryonic fibroblasts endogenously expressing LRRK2 but is amenable to any other cultured cell type as well. For complete details on the use and execution of this protocol, please refer to Fdez et al.¹ and Lara Ordóñez et al.².

Keywords: Cell Biology; Cell Culture; Cell-based Assays; Microscopy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

A549 Cells
Animals
Cell Line
Centrosome*
Coleoptera*
Humans
Mice
Microscopy, Confocal