title: Laser induced wounding of the plasma membrane and methods to study the repair process.
abstract: Cells are constantly exposed to agents that can trigger the perforation of their plasma membrane. This damage occurs naturally, and the frequency and intensity depends on how much cells are exposed to damaging threats. The following protocol is a simple and powerful method to damage the plasma membrane using laser ablation. It allows the induction of a single and localized wound at the plasma membrane of cultured cells, which can be followed with fast time-lapse imaging. The first part of the protocol describes simple cell culture techniques and the material ideal to make the experiments. A second part of the protocol gives advice about the procedures to make effective wounds in cells while ensuring a good survival rate. We also propose different ways to follow the opening and closure of the plasma membrane. Finally, we describe the procedure to efficiently analyze the data acquired after single cell photodamage to characterize the wounding process.
authors: Institut Curie, Paris Cedex 05, France; CNRS UMR, Paris Cedex 05, France.
authors: Ana J
authors: AJ
authors: Jimenez
authors: Institut Curie, Paris Cedex 05, France; CNRS UMR, Paris Cedex 05, France.
authors: Paolo
authors: P
authors: Maiuri
authors: Institut Curie, Paris Cedex 05, France; CNRS UMR, Paris Cedex 05, France.
authors: Julie
authors: J
authors: Lafaurie-Janvore
authors: Institut Curie, Paris Cedex 05, France; CNRS UMR, Paris Cedex 05, France.
authors: Franck
authors: F
authors: Perez
authors: Institut Curie, Paris Cedex 05, France; CNRS UMR, Paris Cedex 05, France.
authors: Matthieu
authors: M
authors: Piel
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000473:pathology*; D003584:Cytological Techniques / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007834:Lasers; D060328:Magnetic Phenomena; D012984:Software; D014945:Wound Healing; D014981:Xenopus
keywords: Laser ablation; Plasma membrane repair; Plasma membrane wounding; Quantitative assay