Cell death is measured using the MTT analysis. This assay is based on the ability of mitochondrial dehydrogenases to oxidize thiazolyl blue (MTT), a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylterazolium bromide), to an insoluble blue formazan product. The cells are incubated with the MTT reagent (120 μg/mL) at 37°C for 2 h. After the supernatant is removed, 400 μL of 0.04mol/LHCl in isopropanol is added to each well, and the optical density of the solution is read at 590 nm in an enzyme-linked immunosorbent assay plate reader. As the generation of the blue product is proportional to the dehydrogenase activity, a decrease in the absorbance at 590 nm provides a direct measurement of the number of viable cells. To determine the contribution of the PI3K pathway to inhibition of apoptosis, some cell populations are pretreated with 50 μM?LY294002, a PI3K inhibitor. Following this pretreatment, cell death is determined as described above.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice pharyngeal aspiration is performed as described. Animals are anesthetized with a mixture of ketamine and xylazine (45 mg/kg and 8 mg/kg, i.p., respectively). Test solution (30 μL) containing LPS (1.5 mg/kg) is placed posterior in the throat and aspirated into the lungs. Control mice are administrated sterile saline (0.9% NaCl). Animals are administered with Arg-Gly-Asp-Ser or RGES peptide (1, 2.5 or 5 mg/kg, i.p.) once one hour before LPS treatment and sacrificed 4 h post-LPS. Animals are also administered Arg-Gly-Asp-Ser or RGES peptide (5 mg/kg, i.p.) once at different time points (1 h before or 2 h after LPS treatment) and sacrificed 24 h post-LPS. In addition, animals are administered with αvβ3-blocking mAbs, anti-αv, or anti-β3?(5 mg/kg, i.p.) once 1 h before and sacrificed 4 h post-LPS. Animals administered with these mAbs 2 h after LPS treatment are sacrificed 24 h post-LPS

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Grigoriou V, et al. Apoptosis and survival of osteoblast-like cells are regulated by surface attachment. J Biol Chem. 2005 Jan 21;280(3):1733-9.  [Content Brief]

  [2]. Moon C, et al. Synthetic RGDS peptide attenuates lipopolysaccharide-induced pulmonary inflammation by inhibiting integrin signaled MAP kinase pathways. Respir Res. 2009 Mar 9;10:18.  [Content Brief]

  [3]. Yin X, et al. Synthetic RGDS peptide attenuated lipopolysaccharide/D-galactosamine-induced fulminant hepatic failure in mice. J Gastroenterol Hepatol. 2014 Jun;29(6):1308-15.  [Content Brief]

  [4]. Aguzzi MS, et al. Intracellular targets of RGDS peptide in melanoma cells. Mol Cancer. 2010 Apr 22;9:84.  [Content Brief]