Proteome profiler human angiogenesis array

The protein array was detected through Proteome Profiler Human Angiogenesis Array (R&D, ARY007). A total of 2.0 ml of array buffer 7 was added to the reaction plate and the reaction film was placed in it until the blue dye on the film disappeared, then incubated on a shaker for 1 h. While the membranes are blocking, prepare samples by adding up to 1.0 ml of each sample to 0.5 ml of Array Buffer 4 in separate tubes. Adjust to a final volume of 1.5 ml with Array Buffer 5 as necessary. A recombinant detection antibody factor (15 μL) was added to each sample, mixed well, and incubated at room temperature for 1 h. Array buffer 7 was removed and the sample/antibody mixture was added. Samples were covered and incubated overnight on a shaker at 2–8°C. The membrane was removed and placed in 1× of wash buffer in a single plastic container of 20 ml for 10 min and repeated twice. A total of 2.0 ml of streptavidin-HRP diluted with buffer five was added into each well, covered with a lid, and incubated on a shaker at room temperature for 30 min. Membranes were washed for 10 min three times, then drained. A total of 1.0 ml of the prepared chemical reagent mixture was evenly dropped on each membrane and incubated for 1 min at room temperature before radiological automatic imaging of the membrane.

Protein contents of MSC-EVs and MCS-CM samples were measured using a BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA). The expression of angiogenic protein was analyzed using Proteome Profiler Human Angiogenesis Array (R&D Systems, catalogue number ARY007). Briefly, MCS-EVs and MCS-CM were mixed with the biotinylated detection antibody cocktail and incubated with nitrocellulose membranes containing spotted specific antibodies. Following adsorption of the biotinylated-antibody–antigen complexes, membrane was washed and further incubated with Streptavidin-HRP for quantification. Cytokine detection was performed using a ChemiDoc XRS+ System (BioRad, California, CA, USA). The signal density of each blot was quantified by dot blot analysis on NIH ImageJ analysis software 1.8.0. The level of 3 pro-angiogenic proteins was measured with a Quantikine ELISA kit (R&D systems): IL8 (D8000C), MCP-1 (DCP00) and CXCL16 (DCX160), following the manufacturer’s instructions.

We used the Proteome Profiler Human Angiogenesis Array (ARY007, R&D Systems, USA) for a qualitative comparison of 55 angiogenic cytokines among the different CM. The culture supernatants from each sample were incubated with the reconstituted detection antibody cocktail. The 55 different angiogenesis antibodies were spotted in duplicate onto the array membrane and incubated with the detection antibody mixture overnight at 4 °C. The membrane was then incubated in streptavidin-horseradish peroxidase conjugate, followed by a chemiluminescent detection reagent. The membrane was scanned using a chemiluminescence detection apparatus, and profiles of mean spot pixel density were created. The level of expression of each cytokine is directly proportional to the luminescence intensity. Data are reported as the mean pixel density of each cytokine. The experiment was performed twice in duplicate.

Conditioned media from differentiated THP-1 either cultured alone or cultured with differentiated primary human breast adipocytes were analyzed using the Proteome Profiler Human Angiogenesis Array (R&D Systems, Minneapolis, MN, USA). Conditioned media from each sample was run on an individual array membrane, which was scanned using a Bio-Rad ChemiDoc XRS+ gel documentation system and measured using Bio-Rad Image Lab software. Intensities were normalized to reference spots on each membrane.