Measuring Cellular COX Activity and VEGF Secretion

Cellular COX activity was measured by stimulation with 50 μM ¹⁴C-arachidonic acid (30 s at 37°C) followed by quantification of radiolabeled prostaglandin products, as described [62 (link)]. The percentage of total products was expressed per 10⁶ cells. Cellular secretion of VEGF into serum-free medium with and without stimulation with arachidonic acid (20 μM) for 24 hours was measured using a human VEGF ELISA plate according to manufacturer's instructions (Signosis Inc, Santa Clara, CA). Sulfurhodamine B (SRB) growth assays were performed as previously described [31 (link), 69 (link)]. Effects on cell growth were measured 72 hours after plating of cells. Clonogenic assays were performed and quantified using the GelCount System (Oxford Optronix, UK) in the DHSR at VUMC [69 (link)]. Cell invasion assays were performed using low basement membrane cell invasion inserts according to manufacturer's instructions (Trevigen, Gaithersburg, MD). Standard 10% FBS growth medium was used as a chemoattractant in the lower chamber for cells grown on the inserts. Following invasion, cells on the side of the insert facing the lower chamber were stained with 1% crystal violet, and the insert carefully mounted on a microscope slide for quantification of invading cells. 5 fields per insert were counted using a 5x objective lens.