Ebm 2

Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial cell growth basal medium-2 (EBM-2; Lonza, Walkersville, MD) supplemented with SingleQuots^TM Supplements and Growth Factors (Lonza; USA, MD). Matrigel (Corning, Flintshire, UK) was diluted with serum-free EBM-2 medium and used to coat in 96-well plates at 37 °C for 1 h. HUVECs were seeded at a density of 1 × 10⁴ cells per well of a 96-well plate in EBM-2 or conditional medium. The number of tube formations was imaged and analyzed using phase contrast microscopy and ImageJ 1.53e software (National Institutes of Health).

To compare pro-angiogenic properties of PBMC^sec, a tube formation assay was performed with human umbilical vein endothelial cells (HUVECs, passage 6) (Fig. 1c). Cells were cultured in endothelial cell growth basal medium-2 (EBM-2; Lonza Group AG, Basel, Switzerland) supplemented with endothelial cell growth medium-2 (EGM-2; BulletKit, Lonza). Prior to the tube formation assay, cells were maintained in EBM-2 containing 2% (vol/vol) heat-inactivated fetal bovine serum (Lonza) overnight and starved in basal EBM-2 for 4 h. Cells were seeded on growth factor-reduced Matrigel Matrix (Corning Inc. Life Sciences, Tewksbury, MA, USA) in μ-slides Angiogenesis (ibidi GmbH, Graefelfing, Germany) at a density of 10⁴ cells/cm² and stimulated with the supernatant obtained from 4 × 10⁶ PBMCs for 3 h. Micrographs were acquired by an inverted phase-contrast microscope (CKX41 Olympus Corporation; Tokyo, Japan) equipped with a × 10 objective (CAch N, 10x/0.25 PhP; Olympus) using a SC30 camera (Olympus) and cellSens Entry software (version 1.8; Olympus). Tubule formation was quantified by the Angiogenesis Analyzer plugin of ImageJ using default settings [37 ].