Matrigel growth factor reduced basement

Aliquots (100 μl) of Matrigel® Growth Factor Reduced (GFR) Basement (Catalog # 354230; Corning, USA) were plated into individual wells of 96-well tissue culture plates (Becton Dickinson) and allowed to polymerize overnight at 37 °C in a cell culture incubator. In the tube formation experiments, three treatment groups were used. Group one consisted of HUVECs cultured alone. Group two consisted of HUVECs cultured with 100 mM glucose. Group three consisted of HUVECs cultured with 100 mM glucose and 25% CMpMSC. Group four consisted of HUVECs cultured with 100 mM glucose and pMSCs. Varying pMSC:HUVEC ratios (1:30, 1:6, and 1:4) were used.
HUVECs were seeded at a density of 3 × 10⁴ cells per well in a complete endothelial cell growth culture medium on the polymerized Matrigel. Following 14 h, the tube network formed was observed under an inverted Nikon ECLIPSE Ti U microscope (Nikon, Japan). Photomicrographs were recorded using a Nikon DS-Qi1 camera and data were analysed with the software (Nikon, Japan). Experiments were carried out in triplicate and repeated as already described.

Aliquots (100 μL) of Matrigel® Growth Factor Reduced (GFR) Basement (catalogue number 354230, Corning, USA) were plated into individual wells of 96-well tissue culture plates (Becton Dickinson) and allowed to polymerize overnight at 37°C in a cell culture incubator. In-DBMSC and pre-DBMSC (group 2), pre-DBMSC (group 3) and untreated DBMSCs (group 1) prepared as described above were then seeded at a density of 3 × 10⁴ per well in DBMSC culture medium supplemented with 50 ng/mL vascular endothelial growth factor (VEGF, R&D Systems) on the polymerized Matrigel. HUVEC (3 × 10⁴ per well) cultured without DBMSCs were used as a positive control, or the HUVEC were cultured with 50 or 100 μM H₂O₂, or with the three groups of DBMSCs. Varying DBMSC: HUVEC ratios (1 : 30, 1 : 6, and 1 : 4) were used. The concentrations of H₂O₂ used for this assay had no effect on the viability of HUVEC as we previously published [22 (link)]. Following 14 h of incubation, the tube network was formed and was observed under an inverted Nikon ECLIPSE Ti U microscope (Nikon, Japan), and photomicrographs were recorded using a Nikon DS-Qi1 camera and data were analyzed with Software (Nikon, Japan). Experiments were carried out in triplicate and repeated as described above.