Separation of BSMCs and VSMCs from the lung using the bi-fluorescent <i>αSMA-hrGFP;NG2-DsRed</i> mouse.

Abstract

<p>(<b>A</b>) Expression pattern of hrGFP and DsRed in BSMCs and VSMCs in the lung of an <i>αSMA-hrGFP;NG2-DsRed</i> mouse. Both BSMCs and VSMCs are hrGFP⁺. BSMCs are DsRed⁻ whereas VSMCs are DsRed⁺. Asterisk (*) indicates the blood vessel in the lung the airway. Arrows (>) indicate the airway. Scale bar: 20 µm. (<b>B</b>) Algorithm for bronchial and vascular smooth muscle cell identification by flow cytometry. CD31⁺ endothelial cells and CD45⁺ immune cells were separated from dissociated lung preparation (left panel) followed by evaluation of hrGFP and DsRed distribution within CD31⁻CD45⁻ population (middle panel). CD31⁻CD45⁻hrGFP⁺DsRed⁻ population corresponds to BSMCs (pointed by an arrow). CD45⁻CD31⁻hrGFP⁺DsRed⁺ population is enriched in vascular smooth muscle cells (VSMCs). (<b>C</b>) Relative <i>Notch3</i> mRNA expression in isolated singly hrGFP⁺ cells and doubly hrGFP⁺DsRed⁺ cells from lung cell preparation as determined by qPCR followed by normalization to 18S. Data show mean ± SD and are representative of three independent experiments. ***p<0.001.</p