Raybio mouse cytokine antibody array kit

A RayBio Mouse Cytokine Antibody Array kit (Ray Biotech, Inc. Cat: AAM-CUST-8) was used to detect a panel of 46 secreted cytokines and chemokines that stored in resting platelets or released from activated Pten^fl/fl and Pten^fl/flPf4-Cre platelets in response to thrombin, respectively. Prepared platelet purity was routinely detected by flow cytometry with platelet-specific CD41 antibody staining (>99%). The potential contamination of other immune cells in prepared platelets used for these cytokine assays was excluded with leukocyte-specific CD45 antibody staining (<0.05%). The manufacturer’s recommended protocol was used to perform this assay. Cytokine protein profiles were quantified by determining band intensities with National Institutes of Health ImageJ software.

Serum samples were individually obtained by centrifugation of the whole blood at 8000 × g for 15 min twice. Three different p14 and 20wo serum batches (each a pool from 6 animals) were analyzed using the RayBio Mouse Cytokine Antibody Array Kit (Raybiotech, Inc. Georgia, USA) and Proteome Profiler^TM Array/Mouse XL Cytokine Array Kit (R&D Systems, Minneapolis, USA) following manufacturer’s instructions. Membranes were scanned using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY) and analyzed using ImageJ/Fiji software. The results were then normalized using internal controls, and the relative protein abundance in p14 compared to adult mouse serum was represented.