跨细胞
全内反射荧光显微镜
生物物理学
荧光显微镜
胞吐
化学
显微镜
细胞生物学
生物
荧光
膜
细胞
内吞作用
生物化学
光学
病理
医学
物理
作者
Erika Jang,Siavash Ghaffari,Warren L. Lee
出处
期刊:Methods in molecular biology
日期:2022-01-01
卷期号:: 115-124
被引量:1
标识
DOI:10.1007/978-1-0716-2051-9_7
摘要
Apical-to-basal transcytosis by endothelial cells can be visualized and quantified using total internal reflection fluorescence (TIRF) microscopy of the basal membrane. Past techniques to study transcytosis including electron microscopy and transwells have several limitations such as confounding from paracellular leakage, low transfection efficiency, and the largely descriptive nature of electron microscopy. After the addition of a fluorescent ligand to the apical endothelial surface, using TIRF to measure exocytosis at the basal membrane bypasses these issues by studying transcytosis across a single cell of a confluent endothelial monolayer in real time. A major benefit of TIRF is that only a small volume of the cell is illuminated, thus greatly reducing background noise from the overlying cytosol in the images. This protocol outlines the steps to image and quantify exocytosis of apically applied fluorophore-tagged low-density lipoprotein (LDL) using TIRF microscopy and MATLAB. A similar approach can be used to study endothelial transcytosis of other ligands such as albumin or high-density lipoprotein.
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