Proteome profiler antibody arrays human xl cytokine array kit

The concentration of 105 inflammation-related plasma proteins was quantified in 11 head and neck cancer patients before and after RT by Proteome Profiler Antibody Arrays (Human XL Cytokine Array Kit, R&D Systems, Minneapolis, MN, USA). 350 µL plasma samples were loaded on each array membrane, followed by incubations with biotinylated detection antibody cocktails and with streptavidin-HRP. Results were visualized with chemiluminescence detection reagents and exposed on X-ray films (CL-XPosure Film, Thermo Scientific, Rockford, United States). Evaluation was performed with ImageJ software.
Proteins showing statistically significant changes on the protein array were analyzed by ELISA. The following ELISA reactions were performed according to the manufacturer’s instructions: BAFF, adiponectin/Acrp30, CXCL5/ENA-78, ApoA1, CD14, TFF3, endoglin/CD105 and complement component C5/C5a. All ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

The radio-immunoprecipitation assay (RIPA; Rockland Immunochemicals, PA, USA) buffer was used to lyse the same numbers of EVs (1 × 10¹⁰ particles). The lysed EV solutions were placed onto the NC membrane of the Proteome Profiler™ Antibody Arrays Human XL Cytokine Array Kit (R&D Systems, MN, USA). After developing the NC membrane with ChemiDoc™ XRS+, the intensities of the cytokine array were quantified using ImageLab software. For data analysis, the average intensity of cytokine array was exported with expression of cytokine array intensities in logarithm base 2. The PANTHER (Protein Analysis THrough Evolutionary Relationships; http://www.pantherdb.org) was utilized to analyze the representative proteins from the cytokine array. The gene ontology (GO) and KEGG pathway were analyzed using the DAVID (Database for annotation, visualization and integrated discovery; https://david.ncifcrf.gov/) for prediction of functionalities.