Egm 2 endothelial singlequots kit

For endothelial differentiation, hASCs were seeded at 100 k cell density per 100 µL of 0.125% NFC hydrogel diluted in endothelial cell growth medium (EBM-2 Basal Medium supplemented with EGM-2 Endothelial SingleQuots Kit, Lonza), on low adhesion 96-well inertGrade BRAND plates^®, and 120 µL of the media was pipetted on top of the hydrogel. Cultures were kept for 21 days with a medium change twice a week. Some of the cultures were stimulated with 100 ng/mL of recombinant human Angiopoietin-1 (Ang-1) (Cat. #130–06; Peprotech, London, UK) upon seeding and during medium changes.

Donor ASCs were used for experiments between passages three to six and cells isolated from individual donors served as replicates (n numbers refer to the number of donors, i.e., the number of repeats of separate experiments with cells from different donors). Human ASCs were cultured in MEM α GlutaMAX™ Supplement medium (MEM-α, Cat. #32571036; Gibco, UK) supplemented with 5% (v/v) of human serum (Cat. #H4522; Sigma-Aldrich).
HUVECs were used for experiments between passages four to six. Cells were cultured in EBM-2 Basal Medium (Cat. #CC-3156; Lonza) supplemented with EGM-2 Endothelial SingleQuots Kit (Cat. #CC-4176; Lonza) or in Endothelial Cell Growth Basal Medium 2 (Cat. #C-22216; PromoCell) supplemented with Growth Medium 2 Supplement Mix (Cat. #C-39216; PromoCell).
Cell culturing was performed at +37 °C in a humified cell culture incubator with 5% CO2. Cell morphology and growth were evaluated with Leica DM IL LED microscope (Leica Microsystems, Wetzlar, Germany).

Hyclone (Logan, UT, USA) provided Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium, and fetal bovine serum (FBS). We obtained EBM-2 medium and EGM-2 Endothelial SingleQuots Kit from Lonza (Basel, Switzerland) and 2-mercaptoethanol from Gibco (Grand Island, NY, USA). We purchased recombinant human TNF-α from BD Pharmingen (San Diego, CA, USA) and recombinant mouse TNF-α from NKMAX (Seongnam-si, Gyeonggi-do, Korea). Sigma-Aldrich (St. Louis, MO, USA) provided 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and analytical-grade reference standards (methyl gallate, catechin hydrate, paeoniflorin, benzoic acid, and paeonol). We used horseradish peroxidase (HRP)-conjugated pierce goat antirabbit IgG (H+L), pierce goat antimouse IgG (H+L), and Alexa Fluor 488 and 594 goat antirabbit IgG (H+L) for the secondary antibodies, purchasing these from Thermo Fisher Scientific Inc. (Eugene, OR, USA). The PLE reference standards, oxypaeoflorin, benzoyl paeoniflorin, and albiflorin, were purchased from Ensol Biosciences Inc. (Daejeon, Korea), Biosynth Carbosynth (San Diego, CA, USA), and FUJIFILM Wako Pure Chemical Corporation (Richmond, VA, USA).