Isolation of endothelial cells was performed according to the method of Sobczak et al. [17] with the following modifications: livers were harvested from adult mice, the liver digest was purified by removal of macrophages with Dynabeads (Invitrogen, Grand Island, NY) coated with anti-CD16 antibody (BD Bioscience, San Jose, CA) for 20 minutes at room temperature, and positive selection was performed by incubation with anti-CD31 (BD Bioscience) coated Dynabeads for one hour at 4°C.
Endothelial cells were grown to 80% confluence in EGM-2 medium (Lonza, Walkersville, MD). They were then washed in PBS and disassociated with Accutase (Invitrogen). Cells were resuspended with in EGM with 5% FBS, and centrifuged at 150 g for 5 minutes. The pellet was then resuspended in FACS buffer (PBS, 5% FBS, 0.1% sodium azide, 2 mM EDTA) at a concentration of 500,000 cells per mL. 100 µL of cells were incubated with periodic agitation with 1 µL of fluorochrome-conjugated anti Flk-1 and PDGFRβ antibodies (BD Bioscience) for 45 minutes at 4°C. The cells were then washed twice with FACS buffer and resuspended in a final volume of 200 µL. Flow cytometry was performed using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).
Endothelial cells were grown to 80% confluence in EGM-2 medium (Lonza, Walkersville, MD). They were then washed in PBS and disassociated with Accutase (Invitrogen). Cells were resuspended with in EGM with 5% FBS, and centrifuged at 150 g for 5 minutes. The pellet was then resuspended in FACS buffer (PBS, 5% FBS, 0.1% sodium azide, 2 mM EDTA) at a concentration of 500,000 cells per mL. 100 µL of cells were incubated with periodic agitation with 1 µL of fluorochrome-conjugated anti Flk-1 and PDGFRβ antibodies (BD Bioscience) for 45 minutes at 4°C. The cells were then washed twice with FACS buffer and resuspended in a final volume of 200 µL. Flow cytometry was performed using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).