Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Summary

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Abstract

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Introduction

The cell membrane of eukaryotic cells consists of a double-layer of protein-studded phospholipids which defines the intra/extracellular environment of the cell and is essential for maintaining cellular homeostasis and cell survival. Cell membrane injuries arising from mechanical or chemical insults are commonplace in various mammalian cell types, including skeletal and cardiac muscle, stomach, and lung cells^1,2,3,4. In addition to injuries consequent to daily physiological function, cell membranes can also be damaged by environmental insults....

Protocol

Human fibroblast cells used in this study were used with approval from the Human Ethics Committee of the Faculty of Medicine and Dentistry at the University of Alberta.

1. Transfection of cells with full-length DYSF plasmid

1. Culture fibroblast cells in a T225 flask containing 40 mL of growth media - 36 mL Dulbecco's Modified Eagle Medium (DMEM), 4 mL of fetal bovine serum (FBS), and 20 µL of penicillin/streptomycin (P/S) - in a CO₂ incubator at 37°C.
   NOTE: Cells should be cultured to approximately 70-80% confluency and should not be allowed to become completely confluent.
2. To prepare cells....

Results

Healthy human fibroblasts, non-treated dysferlinopathy patient fibroblasts, and patient fibroblasts transfected with a plasmid containing the full-length dysferlin sequence were subjected to two-photon laser wounding to assess membrane resealing ability in real time. Healthy human fibroblast cells displayed low levels of FM 4-64 fluorescence activation following laser wounding and non-treated patient fibroblasts exhibited a high degree of relative fluorescence intensity following injury (.......

Discussion

Two-photon laser wounding of the cell membrane is a precise and versatile technique for assessing the dynamics of membrane resealing in vitro. In this article, we described a protocol for determining cell membrane resealing ability in dysferlinopathy patient cells using the two-photon laser wounding assay. Our results show that dysferlinopathy patient cells are defective in cell membrane resealing, which is consistent with findings by other researchers and which further highlights the striking similarities in ce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Thermo Fisher	11320033
Fetal Bovine Serum	Sigma-Aldrich	F1051
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher	15140122
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher	25300062
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D8537
35mm collagen-coated glass-bottom dishes	MatTek	P53GCOL-1.5-14-C
Serum-deprived media	Thermo Fisher	31985070
Transfection reagent	Thermo Fisher	15338100
FM 4-64 Dye	Invitrogen	T13320
Tyrode’s Salts Solution	Sigma-Aldrich	T2397
Confocal laser scanning microscope	Carl Zeiss	NA
Chameleon Two-photon laser	Coherent	NA