Recombinant sparc

Human coronary artery ECs (CAECs, Promocell) were seeded on the top of Matrigel (Corning® Matrigel® Growth Factor Reduced Basement Membrane Matrix) (4,000 cells/well) using Angiogenesis μ-Slides (IBIDI, UK). APC-CM was diluted 1:1 with fresh EBM2 medium. EBM2 was used as control. Recombinant SPARC (Peprotech, UK) was diluted in EBM2. Images were snapped after 5 hours, and the total tube length per imaging field was measured.

To test the capacity of the APC secretome to induce the migration of CSCs, HUVECs and lung fibroblasts (60,000 cells) were seeded in 24-well plates on a 6.5-mm Transwell with 8-μm pore polycarbonate membrane insert (Corning, UK) in serum-free EGM-2. In the bottom of the wells, 0.5 mL of APC-CM was added. Complete EGM-2 was used as reference control. Cells were incubated for 17 hours at 37°C. At the end of this period, the membrane inserts were washed with PBS, fixed with icecold methanol for 15 minutes at room temperature, stained with DAPI, and mounted on a slide. Membranes were analyzed with an epifluorescence microscope at x200 magnification. Migrated cells were expressed as a percentage. All experiments were performed in duplicate. For experiments with SPARC-silenced CM, we used the fluorometric CytoSelect 96-Well Cell Migration Assay (CBA-106, Cell Biolabs, UK), according to the manufacturer's instructions, with experiments being performed in triplicates. Recombinant SPARC (Peprotech, UK) was diluted in EBM2 to the indicated concentration.

Proliferation was measured using either a BrdU colorimetric (Roche, Switzerland) or an EdU fluorescent (ThermoFisher) kit. Apoptosis was assessed with a CaspaseGlo 3/7 (Promega, UK) assay. Cell viability/apoptosis was assessed using a Calcein-AM/EthDIII kit (Biotium, UK), all according to the manufacturer's instructions. Cells were exposed for 16-24 hours to the experimental conditions before the functional readouts. Experiments were performed in 96-well plates in triplicates. Recombinant SPARC (Peprotech, UK) was diluted in EBM2.