24-Well Cell Invasion Assays, Basement Membrane

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	Starve cells for 24 hours. Incubate for 24 hours at 37ºC in 5% CO2 atmosphere

Protocol tips
Starve cells for 24 hours. Incubate for 24 hours at 37ºC in 5% CO2 atmosphere

Publication protocol

Cell invasion was determined using the CytoSelect cell invasion assay (Cell Biolabs, San Diego, CA) according to the manufacturer's instructions. Briefly, cells were serum-starved for 24 h. Basement membranes of Boyden chambers were rehydrated with 300 μL serum-free medium, and 1 × 106 cells in appropriate serum-free medium were seeded into the upper chamber containing antagonists or agonist (isoproterenol, a non-selective agonist) for β adrenoceptors. Bottom wells were filled with medium supplemented with 10% FBS. Propranolol concentrations were 150, 50, and 15 µM for MCF7, HT-29, and HepG2 cells, respectively. Atenolol concentrations were 600 µM MCF7 and HT-29, and 2400 µM for HepG2 cells. About 90, 20, and 10 µM ICI118,551 were applied to MCF7, HT-29, and HepG2 cells, respectively. Ten µM isoproterenol (Sigma) were applied (Lung et al., 2005 Lung HL, Shan SW, Tsang D, Leung KN. (2005). Tumor necrosis factor-alpha mediates the proliferation of rat C6 glioma cells via beta-adrenergic receptors. J Neuroimmunol 166:102–12Yang et al., 2009 Yang EV, Kim SJ, Donovan EL, et al. (2009). Norepinephrine upregulates VEGF, IL-8, and IL-6 expression in human melanoma tumor cell lines: Implications for stress-related enhancement of tumor progression. Brain Behav Immun 23:267–75


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