Isolation and Characterization of Prdx6-null Pulmonary Microvascular Endothelial Cells

Pulmonary microvascular endothelial cells (PMVEC) were isolated from the lungs of Prdx6-null mice as described previously [28] (link). The endothelial phenotype was routinely confirmed by fluorescence microscopy to determine cellular uptake of acetylated low-density lipoprotein labeled with 1,1′–dioctadecyl-3,3,3′,3′–tetramethylindocarbocyanine perchlorate (DiI-AcLDL) and by immunostaining with mouse monoclonal antibodies for PECAM-1 (Santa Cruz Biotech, Santa Cruz, CA) and vascular endothelial cadherin (VE-cadherin) (Abcam, Cambridge, MA) [28] (link). Prdx6 null PMVEC were infected by adding lentivirus prepared using primers as described previously to cells in growth media containing polybrene (∼8 μg/ml) [16] (link), [19] (link). Cells were studied at 72–96 h post-transfection when expression of infected proteins was maximal. Empty HMD lentiviral transfer plasmid was used as a negative control.