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

Download

Copyright Disclaimer:
This site does not store any files on its server. We only index and link to content provided by other sites. Please contact the content providers to delete copyright contents if any and email us, we'll remove relevant links or contents immediately.