μ angiogenesis slides

The tube formation assay measures the ability of endothelial cells to form tubes (capillary-like structures). In order to perform this assay, 10 μL of Geltrex^® (Invitrogen, Thermo Fisher Scientific) was added to the wells of μ-slides Angiogenesis^® (IBIDI, Martinsried, Germany) and allowed to solidify at 37 °C for 30 min. After the gel solidified, SVEC4-10 cells (1 × 10⁴) were added in 50 μL of DMEM supplemented with 10% FBS. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ in air for 5 h and then analyzed. The following parameters were used for quantification [62 (link),63 (link),64 (link),65 (link)]:
Tubes were considered to be tubular structures that extend from one branching point to another branching point or to a loose end. Loops are enclosed (or almost enclosed) areas inside the tubes that fulfill roundness conditions.
The images were acquired using a Nikon Eclipse TE2000-U microscope (Nikon USA, Melville, NY, USA) equipped with a 10× objective. The measurements are expressed in pixels (800 × 600) where 1 pixel equals 0.069 mm². WimTube (Wimasis Image Analysis, Munich, Germany; www.wimasis.com/en/products/13/WimTube) was used to analyze and quantify tube formation.

The angiogenic capacity of cells was assessed with a tube forming assay as described [28 (link)], using reduced growth factor-Matrigel (BD Biosciences) in μ-Slides Angiogenesis (Ibidi). In brief, the optimal number of cells was seeded onto Matrigel-coated Ibidi slides and photographed at the indicated time. For treatment with nor-NOHA, U-251 MG-ARG2 was either mock-treated or treated with 1 mM or 3 mM nor-NOHA for 5 days before seeding onto Matrigel and photographed.

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 ].