Mouse vegf quantikine elisa

Whole eyes were homogenized in PBS and sonicated. After centrifugation, supernatants containing most soluble proteins were collected. VEGF protein levels in the supernatant were determined using an ELISA kit (Quantikine ELISA mouse VEGF; R&D Systems, Minneapolis, MN, USA). Absorbance values were obtained at 450–570 nm using an Emax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

Posterior eyecup without conjunctiva, sclera, and muscle tissue was homogenized in RIPA buffer with protease inhibitor. After centrifugation, supernatants containing the soluble proteins were collected. VEGF protein levels in the supernatant (40 µg protein/well) were determined using an ELISA kit per the manufacturer’s instructions (Quantikine ELISA mouse VEGF; R&D Systems, Minneapolis, MN, USA). Absorbance was measured at 450–570 nm using a SpectraMax iD5 (Molecular Devices, Sunnyvale, CA, USA). Data are represented as pg/mL.

B16F10-sTAC tumors were flash frozen in liquid nitrogen, ground and homogenized in PBS containing 0.5 μM dithiothreitol, 0.5 μM Pefabloc and 8 μg/mL leupeptin. Samples were analyzed for monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6). IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) using the Mouse Inflammation Cytometric Bead Array reagents (BD Biosciences, San Jose, CA) and flow cytometry as per the manufacturer's protocol. VEGF cytokine levels were analyzed using the mouse VEGF Quantikine ELISA (R&D systems) as per the manufacturer's instructions.