HUVECs were isolated from anonymous umbilical veins, as described before72 (link). HUVECs were subcultured using a trypsin/EDTA-reagent pack (Lonza) and maintained in 5% fetal bovine serum (FBS)-containing EC growth medium (Sciencell). Cells were treated or not for 24 h with 1 μg/mL ALK1-Fc (R&D Systems) in complete EC growth medium containing 5% FBS. Cells were then rinsed with PBS and processed for RNA extraction and RNA-Seq as described above. For WB analyses, cells were processed as before73 (link) with the following modifications. Cells were solubilized in RIPA buffer (EMD Millipore) supplemented with 1× Complete protease inhibitor mixture (Roche Applied Science). 5–20 μg of proteins (depending on the primary Ab used) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were then probed with Abs directed against phospho-Smad1/5/8 (Cell Signaling Technology), total Smad5 (Cell Signaling Technology), ID1 (BioCheck), ANG2 (Santa Cruz Biotechnology), and actin (BD Transduction Laboratories). A standard ECL detection procedure was then used.
Ec growth medium
Manufactured by ScienCell
Sourced in United States
EC growth medium is a specialized cell culture medium designed to support the growth and proliferation of endothelial cells. It provides the necessary nutrients and growth factors to maintain endothelial cell viability and function in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
Alk1 fc, by R&D Systems (1 mentions)
Trypsin edta reagent pack, by Lonza (1 mentions)
Ang 2, by Santa Cruz Biotechnology (1 mentions)
Total smad5, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Primary huvecs, by ScienCell (1 mentions)
3 protocols using ec growth medium
1
Isolation and Analysis of HUVECs
2
HUVEC Propagation and Culture
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Primary HUVECs and EC growth medium were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Culture flasks were coated with poly-l-lysine before use, and the cells were cultured in EC supplemented with EC growth supplement, 5% FBS and penicillin/streptomycin solution at 37°C in 5% CO2. HUVECS in the 2nd to 5th passages were used in all experiments.
3
Assessing HUVEC Damage Under Hyperglycemia
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Cell Culture and HG Treatment HUVEC cell lines were purchased from Qida Biotechnology (Shanghai, China) and then cultured in an EC growth medium (ScienCell, USA), including 5% fetal bovine serum and 1% EC growth supplements. The 3rd-6th passages of cultured HUVECs were used for the following experiments. To find the optimal concentration of HG that caused the HUVECs' damage, we mimicked a model of hyperglycemia conditions by treating HU-VECs with 10, 30, and 50 mmol/L of HG.
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