Ab213878

V-PLEX, U-PLEX, and R-PLEX assays from the Meso Scale Discovery multispot assay system were used to quantify proteins in aorta tissue culture medium, BMDM culture medium, and plasma. Customized panels were used according to the manufacturer’s instructions. V-PLEX Panel 1 (K15048D) includes IL-1β, IL-4, IL-6, IL-10, IL-12, and CXCL1. V-PLEX Panel 2 (K15245D) includes CCL2, CCL3, CXCL2, and CXCL10. CCL5 (K152A2K-1), TGF-β Combo (K15242K-1) and MMP9 (B22ZG-2) were measured by U-PLEX assay. TIMP1 (F22YO-3) was measured by R-PLEX assay. MMP12 was measured by ELISA (ab213878, Abcam) according to the manufacturer’s instructions.

The quantity of IL-33 (Millipore), MMP-12 (ab213878), p16 (ab230131), and TGFβ1 (ab119557, Abcam, Cambridge, UK) in the mouse lung was measured using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol. SASP and inflammatory cytokines were included in a 26 plex Luminex cytokine array (ThermoFisher, cat # EPX260-26088-901) and performed according to the manufacturer’s protocol. Results are normalized to total protein determined using BCA assay (Pierce).

48h after BMSCs transfection with miR151 or miR5100 2 ml of culture medium was collected and frozen. Medium was analyzed using ELISA assay to determine the concentration of MMP12 (ab213878, Abcam). Assay was performed according to the manufacturer’s protocol. 562 nm absorbance was measured using a microplate reader BioTek ELx800 (Agilent) with Gen5 software (Agilent). The values obtained from the medium were excluded from analysis. Three independent experiments were performed in duplicates. The average results for each experiment were shown on graphs.