24-Well Cell Invasion Assays, Basement Membrane

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	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
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar] ). After 24 h incubation (37 °C, 5% CO2), non-invasive cells were removed from the upper chamber and cell invasion was assessed by light microscopy after staining of invaded cells with cell stain solution. For colorimetric quantification of invasion, inserts were then placed in extraction buffer (200 μL, for 10 min), and absorbance (A) at 560 nm was determined after transfer to a 96-well plate (100 μL/well) using an ELISA reader (Biotek Instrument ELx800, Winooski, VT). Changes in cell invasion due to antagonist and isoproterenol applications were expressed as percent relative to untreated control as follows:

All assays were performed as duplicates.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Cell migration / Invasion cell type - HepG2 using 24-Well Cell Invasion Assays, Basement Membrane from Cell Biolabs.

Manufacturer protocol

Download the product protocol from Cell Biolabs for 24-Well Cell Invasion Assays, Basement Membrane below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Cell migration / Invasion cell type - HepG2 using 24-Well Cell Invasion Assays, Basement Membrane from Cell Biolabs. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.