Abstract
This protocol describes how to isolate murine endothelial cells from newborn mice brain and 3-month-old mice lung by modifying the original protocols (Sobczak et al., 2010; Ruck et al., 2014). We have used the protocol to analyze mRNA expression level in brain endothelial cells (Sawaguchi et al., 2017). Isolated lung endothelial cells were expanded in vitro for various downstream experiments such as gene expression analysis and cell-based signaling assay.
Keywords: Endothelial cell, Mouse, Isolation, RT-PCR
Background
This protocol describes experimental procedures for isolation of murine endothelial cells from mouse brain and lung. We used newborn mice for isolation of brain endothelial cells using anti-CD31 antibody. With this protocol, 2 μg of total RNA which is suitable for gene expression analysis can be isolated from brain endothelial cells, although pericytes and astrocytes cannot be totally eliminated. We also used 3-month-old mice lung to isolate lung endothelial cells using anti-CD31 and anti-CD102 antibodies. Isolated lung endothelial cells were expanded in vitro in 6 cm dish and used for various downstream experiments such as gene expression analysis and cell-based signaling assay.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
We thank N. Toida (Nagoya Univ) for technical supports. This protocol was modified from the previously published article (Sawaguchi et al., 2017). This work was supported by Japan Society for the Promotion of Science grants #JP15K15064 to TO and MO, #JP26110709 to TO, #JP26291020 to TO, #JP15K18502 to MO, #JP16J00004 to MO; Takeda Science Foundation to TO; Japan Foundation for Applied Enzymology to TO; YOKOYAMA Foundation for Clinical Pharmacology #YRY-1612 to MO. The authors declare no conflict of interest.
References
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