Proteome profiler human angiogenesis array kit

The Proteome Profiler Array (Human Angiogenesis Kit; R & D Systems Europe, Abingdon UK) was used to recognize site-specific pro-angiogenic elements in the condensed culture media. Cell protein lysates were obtained and detected using the kit. Arrays were prepared and incubated according to manufacturer recommendations. A 30-min incubation at room temperature was performed with the streptavidin-HRP solvent. Membranes were laundered three times, and signals were detected by gel chemiluminescence imaging. ImageJ was used to quantify the pixel intensity in each spot after scanning, adjusting size, and inverting the array films using Adobe Photoshop. After calculation of the mean signal of duplicate spots corresponding to each protein, a clear region of the membrane was subtracted from the background signal.

We used the Proteome Profiler Array (Human Angiogenesis Kit, R&D Systems Europe, Abingdon UK) to identify specific pro-angiogenic factors in the conditioned media. We blocked (array blocking buffer) the Angiogenesis Array membrane for 60 minutes then washed it (1X array wash buffer) before leaving conditioned media to incubate overnight at 4 °C. We pre-treated the media with a 1X lysis buffer provided in the kit. Following incubation, we washed membranes (1X array wash buffer) and incubated streptavidin-HRP solution at room temperature for 30 minutes. We washed membranes three times (1X array wash buffer) and treated them with Lumi Glo and peroxide. We detected signals via Gel Chemiluminescence Imaging - Fusion SL (Vilber Lourmat, Eberhardzell, Baden-Württemberg, Germany). We estimated cytokine concentration by counting spot pixels using Image J software and normalizing means to the negative and positive controls provided by the manufacturer. We conducted one array analysis for single cell-culture media and two for co-culture conditioned media. A factorial ANOVA was used to test cytokine concentration across media.

To characterize the paracrine activity of ASCs, conditioned medium was collected, centrifuged at 2,500 g to remove cell debris, and stored at –70 °C until the measurements were taken. To detect secreted proteins, conditioned medium was analyzed using the Proteome Profiler Human Angiogenesis Array Kit (R&D, USA) and Proteome Profiler Human Protease Array Kit (R&D, USA), according to the manufacturer’s instructions. The data were analyzed using Image Lab™ Software Version 5.0 (Bio-Rad, USA). VEGF-α, TGF-β, IL-6, IL-8, MCP-1, and IGF-1 concentrations in ASC conditioned medium were evaluated using the Human VEGF ELISA Set (Peprotech, USA), Human TGF-β1 DuoSet ELISA (R&D, USA), Human IL-6 ELISA Set (BD, USA), Human IL-8 ELISA Set (BD, USA), Human CCL2/MCP-1 DuoSet (R&D, USA), and Human IGF-1 DuoSet (R&D, USA), according to the manufacturer’s instructions.

Proteome Profiler assays were performed in the plasma from the screening cohort via Proteome Profiler Human Angiogenesis Array Kit (catalog #ARY007, R&D Systems, Inc., Minneapolis, MN, USA) and Proteome Profiler Human Cytokine Array Kit (catalog #ARY005B, R&D Systems) respectively according to the manufacturer's instructions. The array kits were designed to detect 55 angiogenesis-associated proteins and 36 cytokines in a single experiment by utilizing a sandwich immunoassay. Furthermore, pixel densities of the films were analyzed by Image J software (Image J software, 1.52a National Institutes of Health, Bethesda, MD).