Sensolyte 520 mmp 2 assay kit

Saos-2, MG-63 and SJSA-1 human OS cell lines and PK136 hybridoma cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). ETAR shRNA lentiviral particles (sc-39960-V), control shRNA lentiviral particles-A (sc-108080), selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (sc-364437A), anti-ETAR (sc-21193) antibody, anti-matrix metalloproteinase-2 (MMP-2) antibody (sc-53630), anti-Akt (5C10) (sc-81434) and anti-P-Akt (ser473) (sc-101629) antibodies, and a mouse anti-ganglioside GD2 mAb (14G2a) (sc-53831) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A mouse isotype-matched control PK136 mAb for 14G2a were purified from hybridoma culture supernatants using the Hi-Trap Protein G HP column (GE Healthcare Life Sciences, Shanghai, China) according to the manufacturer's protocol. The SensoLyte 520 MMP-2 Assay Kit (71151) was purchased from AnaSpec (Fremont, CA, USA). The QCM ECMatrix 24-well (8 µM) Fluorimetric Cell Invasion Assay kit (ECM554) was purchased from Chemicon (Millipore, Billerica, CA, USA). The ET-1 ELISA kit and the Methlythiazoletetrazolium (MTT) cell viability assay kit was purchased from R&D Systems (Minneapolis, MN, USA). The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA). Selective ETAR antagonist BQ123 was purchased from Sigma (St. Louis, MO, USA).

AA tissues were homogenized using CytoBuster™ protein extraction buffer (Novagen, Merck KGaA, Darmstadt, Germany). Lysate protein concentration was measured using a Qubit Protein Assay Kit and Qubit 2.0 fluorometer (Thermo Fisher Scientific). An equal concentration of total protein was applied to each enzyme-linked immunosorbent assay (ELISA) kit (IL-4, IL-10, IL-1β, IL-6, TGF-β1, TNF-α, TIMP-1, and MCP-1: Invitrogen, Carlsbad CA, USA; TIMP-2: Abcam), and the amount of protein was determined. Endogenous active MMP-2 and MMP-9 in aortic tissues were measured using a SensoLyte 520 MMP-2 assay kit (ANASPEC, Fremont, CA, USA) and a mouse MMP-9 activity assay kit (QuickZyme Bioscience, Leiden, the Netherlands), respectively. An equal concentration of total protein was applied to each assay kit and then detected.

The aorta from the infradiaphragm to the superior mesenteric artery was harvested from AA model mice. The AA tissue was cut into round, 1-mm-thick slices. One slice of AA tissue was plated onto 24-well plates (Transwell; Corning) and then cultured with 1 × 10⁶ M2Ms that were cocultured using cell culture inserts (Transwell; Corning) (n = 4 mice). These were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air for 7 days without media change. Monoculture (a slice of AA tissue alone) was incubated in the same manner as control (n = 4 mice). At 7 days, the incubated AA tissue was retrieved and endogenous active MMP-2 and MMP-9 were measured using a SensoLyte 520 MMP-2 assay kit (ANASPEC) and a mouse MMP-9 activity assay kit (QuickZyme Bioscience), respectively.