Pbmn z

Isolated LEC and BEC populations were retrovirally infected with fluorescent proteins to visualize network formation. The cDNA encoding pLV-EGFP, pLV-mCherry, and pBMN-Z was purchased from Addgene (Cambridge, MA, USA). The pcDNA3-EYFP-HIS was purchased from Invitrogen (ThermoFisher, Waltham, MA, USA). The eGFP and mCherry were subcloned into the pBMN backbone after digestion with BamHI and SalI. The EYFP-HIS was subcloned into pBMN after digestion with BamHI and EcoRI. Phoenix Ampho cells were a kind gift from Regina Grillari (University of Natural Resources and Life Sciences, Vienna, Austria) and cultured in DMEM 10% FCS. Virus particle generation was performed by transfecting Phoenix cells at 80% confluency using Lipofectamine 2000 or TurboFect (ThermoFisher, Waltham, MA, USA), following the manufacturer’s instructions. The supernatants containing virus particles were transferred onto 80% confluent LEC and BEC, and virus particles were centrifuged onto the cells at 800 × g for 60 min. The supernatant was then removed, and the cells were incubated overnight in EGM-2. Retrovirally infected cells were then expanded in new flasks and used for subsequent experiments.

EGFP and mCherry in pLV vectors and pBMN-Z were purchased from Addgene (Cambridge, MA, USA). pcDNA3-EYFP-HIS was purchased from Invitrogen (ThermoFisher, Waltham, MA, USA). EGFP and mCherry were subcloned into pBMN after digestion with BamHI and SalI. EYFP-HIS was subcloned into pBMN after digestion with BamHI and EcoRI. Phoenix ampho cells were a kind gift of Regina Grillari (University of Natural Resources and Life Sciences, Vienna, Austria) and cultured in DMEM 10% FCS. Virus particle generation was performed by transfecting Phoenix ampho cells at 80% confluency using lipofectamine 2000 or TurboFect (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. Supernatant containing virus particles was mixed 50:50 with full medium and transferred onto 80% confluent HUVEC and incubated overnight. YFP-HUVEC, GFP-HUVEC and mCherry-HUVEC were then expanded in new flasks and used for subsequent experiments. The label was chosen based on the prerequisites of the respective experiment (e.g. complementary stainings with fluorescent dyes).