Matrigel assay

The BD BioCoat Angiogenesis system - endothelial cell tube formation - Matrigel assay (BD Biosciences, MA USA) was used to assess the anti-angiogenic activity of the T3, T7 and LF-15 peptides. Endothelial cells were seeded onto a 96 well plate at 4×10⁵ cells/ml in F-12 media with ECGS containing 10% FBS, with 100 ng/ml angiopoietin-1 (R&D systems, Minneapolis, USA) 300 ng/ml ephrine B2 (R&D systems) and 100 ng/ml VEGF (R&D systems). The T3, T7 and LF-15 peptides or corresponding vehicle controls, were added to triplicate wells at a concentration of 4.5µM. After 18 hours incubation at 37°C in 5% CO₂, tube formation was visualised using an inverted light microscope. Images were taken using a digital camera (Olympus CAMEDIA C-4000) and the total number of tubes per well were counted manually.

The ability of IM-HBMEC to migrate and generate capillary-like structures was assessed by using the Matrigel assay (BD, Franklin Lakes, NJ, USA). Fifty microliters of growth factor reduced Matrigel matrix were added to a 96-well plate and allowed to solidify at 37 °C for 30 min. CMs harvested from CAS-1 and U87MG 48 h after transfection were centrifuged at 1000× g for 5 min, filtered with a 0.2 μm filter and stored at −80 °C until use. For all conditions, CMs were used at 1:1 dilution with serum and endothelial cell growth supplement-free HBMEC medium. IM-HBMEC (10⁴ cells/well) were treated with CMs or 25 ng/mL recombinant human VEGF-A (VEGF-A165 isoform) (Peprotech, Rocky Hill, NJ, USA) for 6 h (37 °C, 5% CO₂) to allow the formation of tube-like structures. Photographs at 40× magnification were obtained using an inverted Leica DM IRB microscope equipped with a charge-coupled device (CCD) camera, as previously described [65 (link)]. Tube formation was assessed by measuring the total tube length and counting tubule branch points. The latter can be defined as cell junctions with at least three tubules (sections of the tube structure where three or more tubes converge). Angiogenesis Analyzer tool for ImageJ software (National Institutes of Health) was used for quantifications.

Pipette tips, pre-chilled, were used to draw 50 μL of Matrigel assay (BD Biosciences, Bedford, MA, USA) uniformly across the base of a 96-well plate. Following distribution, 96-well plates were situated in a 37 °C cell culture incubator for 30 ~ 60 min for solidification. Test cells were digested, and cell suspensions were prepared. The cells were then counted and adjusted to 25,000 cells and 200 μL of cell suspension per well was injected into a 96-well plate containing three matrix wells. After 6 h, cells were observed using an inverted light microscope and imaging system. The total branch length of endothelial cells in each group was quantified using vascular analysis software (Angiogenesis analyze). The tube formation assay was repeated thrice. The assay was conducted as previously described^{24 (link)}.

Promotion of in vitro angiogenesis by APCs was assessed using a Matrigel assay (BD Biosciences). HUVECs (Lonza) and APCs were cultured together in a 96-well plate on 70 μL Matrigel in a ratio of 3:1 HUVEC:APC for 6 hr. Total tube length was measured, along with the number of APCs found on nodes and branches. To visualize APCs, the cells were stained with long-term cell tracker VyBrant diI (Life Technologies, UK). DiI was diluted 1:1,000 in PBS and incubated with adherent confluent APCs for 5 min at 37°C and then on ice for a further 15 min in the dark. Cells were then washed with PBS, left to recover for 24 hr, then used for experiments.
To determine the role of Ang-1 in HUVEC network formation, HUVECs were cultured on Matrigel with PC-conditioned media plus a Tie-2 inhibitor (Abcam). In brief, HUVECs in 100 μL EGM-2 were seeded onto 70 μL Matrigel along with either 100 μL serum-free-conditioned media from PCs treated with scrambled sequence or miR-532-5p mimic plus/minus 7.5 μM Tie-2 inhibitor. Cells were incubated for 6 hr, then the total tube length measured.

Tube formation ability was evaluated using a Matrigel assay (BD, USA). Matrigel (9.2 mg/ml) was used at 1:1 dilution with a completed medium. ECs were seeded on Matrigel with VEGF (R&D, CDN) (20 ng/ml) and tofacitinib (1 μM). The concentration of tofacitinib followed what had already been published in similar experimental models [21 (link)–23 ]. After 4 h, the images were acquired using an Olympus BX53 microscope. The total tube length, the total branching length and the number of junctions of each experiment were measured, using NIH ImageJ. Results were expressed as median (range).

Matrigel assay (BD Biosciences, Buccinasco, Italy) was used to assess the cell invasive potential, according to the manufacturer's protocol. Briefly, cells were plated into upper inserts of 24-well plates (at a density of 60×10³) and incubated at 37°C. Bottom chambers were filled with 500 µl of DMEM supplemented with 10% FBS as a chemoattractant or DMEM only as a negative control. After 24 hours, non-invading cells were gently removed from the upper surface of inserts with a cotton swab and invaded cells were methanol fixed and stained with Crystal violet (0.1% in PBS with 20% methanol). The number of cells that invaded the filter was counted using a bright-field microscope. Five randomly selected fields were counted for each filter and experiments were performed in triplicate.