Vascular Hyperpermeability by Unknown

Vascular Hyperpermeability by Unknown

Author:Unknown
Language: eng
Format: epub
ISBN: 9781071634295
Publisher: Springer US


1 Introduction

Visualization of the spatiotemporal nature of proteins in live endothelial cells is a key method for understanding subcellular mechanisms that determine functions such as the control of microvascular permeability. There are a variety of methods for transfection of endothelial cells using chemical agents, viral vectors, and electroporation with vectors encoding fusion proteins [1–4]. We have had reproducible success with a Nucleofector II system (Lonza, Basel Switzerland), which utilizes a combination of electroporation and optimized chemical transfection medium [5–9]. For this system it is important to be able to quickly to maximize the viability of the cells. Each transfection requires 5 × 105 cells, which after transfection are seeded onto a thin glass that allows for optimal optics for microscopic viewing at high power. Before the development of specialized cultureware for live cell imaging, the cells were seeded onto #1 glass coverslips kept in a culture dish. On the day of the imaging study, these coverslips were mounted together with a plastic top to form a chamber [7]. However, the emergence of 35-mm culture dishes with a recessed coverslip located in the center (Fig. 1a) and a corresponding perfusion insert (Fig. 1b) makes an easier and more productive alternative because the chance of accidentally breaking the coverslip during various steps of the protocol is drastically reduced.

Fig. 1 Cultureware-chamber system for live cell imaging. (a) Closeup view of a MatTek 35-mm dish with a recessed glass coverslip in the middle of the dish, on which cells are grown for time-lapse imaging. (b) Chamber insert sold by Warner instruments that fits into the 35-mm plate and allows for superfusion of the cells with warmed medium during live cell imaging experiments



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