Human proteome profiler array kit

Xenograft-derived cell cultures were generated by a 10-day cultivation of liberase-digested tissue in basal conditions. Supernatant (0.5 mL) was taken 2 days after medium renewal from a 500,000 cells cultured and analyzed with human Proteome Profiler Array kit (ARY005B, R&D Systems, Abingdon, UK) following manufacturer’s instructions. ImageJ was used for quantification of the dot blots.

Rag1^−/−FVB/n Xenograft-derived cell cultures were generated by a 10-day cultivation of liberase-digested tissue in basal conditions. Supernatant (0.5 mL) was taken 2 days after medium renewal and analyzed with human Proteome Profiler Array kit (R&D Systems, ARY005B) following manufacturer’s instructions.

L-EV_Mreg were semi-quantitatively evaluated for the presence of 55 angiogenesis related proteins using a human proteome profiler array kit (R&D Systems, Minneapolis, MN, USA) as described in the manufacturer´s protocol. Briefly, isolated L-EV_Mreg samples resuspended in PBS were sonicated for 20 min on ice to release intravesicular proteins and were applied to the array membranes carrying antibodies against the respective pro-angiogenic proteins. After incubation, a cocktail of biotinylated antibodies and HRP-streptavidin was added to the membranes and the signals were visualized by chemiluminescence detection, referring to the manual provided. Photographs of the membranes were taken using the Fusion FX Vilber (Vilber Lourmat, Eberhardzell, Germany) and signal intensities were analyzed employing the ImageJ 1.41 software (NIH), and Vilber Smart imaging (Vilber Lourmat).