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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Isolation of Mouse Coronary Endothelial Cells

Published: July 3rd, 2016

DOI:

10.3791/53985

1Department of Physiology, University of Arizona, Tucson

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

Mouse models of various cardiovascular diseases and metabolic disorders bear physiological and molecular changes which are similar to those found in patients. Furthermore, genetic alteration of mice is a powerful tool that allows us to investigate the pathogenic role of specific genes in the development and progression of diseases. Nowadays, cell-type-specific gene-knock-in or -out mice are easily generated in many laboratories and the measurement of mRNA and protein levels in specific cell types is the first step in determining whether the mice were truly genetically modified.

Endothelium is a thin single layer of endothelial cells (ECs) t....

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The research on mice was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Arizona and was performed according to the National Institute of Health (NIH) guidelines.

NOTE: Sections 1, 2 and 3 should be carried out the day before the experiment as setup.

1. Preparing Solutions

  1. CD31 buffer (50 ml)
    1. Add 50 ml 1× Phosphate Buffer Saline (PBS) to a 50 ml tube. Weigh 0.05 g Bovine Serum Albumin (BSA) and 0.037 g Ethylenediaminetetraacetic acid (EDT.......

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The successful isolation of coronary endothelial cells from a mouse heart typically yields around 104 cells with over 90% purity with a cobblestone shape. For a pure population of endothelial cells, caution should be taken throughout the protocol to ensure a sterile environment free from any contamination. The appearance of elongated cells or enlarged cells within the population indicates contamination with other cell types, smooth muscle cells and fibroblasts. Endothelial cell.......

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The most widely used endothelial cells in research are human umbilical vein endothelial cells (HUVECs) because of their convenience and ease of culturing. However, a lot of research requires the availability of a physiological model attained by the isolation of endothelial cells from other specific organs10. Endothelial cells make up a very minor population of cells in the heart tissue; their isolation and culturing can prove to be very difficult.

There are three critical steps with.......

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This work was supported by the grant from the National Institutes of Health (HL115578 to A. Makino).

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Name Company Catalog Number Comments
BSA Sigma A3311-10G
EDTA Fisher BP120-500
HBSS Fisher SH3026801
Hepes Sigma-Aldrich H4034-500G
Sodium Bicarbonate  Sigma S5761
D-(+)-Glucose  Sigma G7021-1KG
Sheep anti-rat IgG beads  Life Technologies F-410L DynaBeads pan mouse IgG , Thermo Fisher Scientific, cat# 11035
Rat anti-mouse PECAM Antibody  BD Pharmigen 550274 BD Biosciences
IFCS  Fisher SH30072.04
M199 Fisher MT10060-CV
Dispase (Neutral Protease) Worthington Biochemical Corp./Fisher LS02109
Collagenase Type II  Worthington Biochemical Corp./Fisher LS004176
Glycerol  Fisher BP381-1
Igepal Sigma 13021-50ml I3021-50 ml
Phosphatase inhibitor cocktail Sigma P0044-1ml
Protease inhibitor cocktail Sigma P8340-1 ml
Heparin  Sigma H3149-100KU
FBS  Fisher/Mediatech MT35010CV
D-Valine Sigma V1255-5G
EGS BD 356006 Corning
Penicillin/Streptomycin Fisher/Mediatech MT-30-002-Cl MT-30-002-CI

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