Prostaglandin e2 elisa monoclonal

Prostaglandin E2 ELISA-monoclonal (Item No. 514010, Cayman Chemical Company, United States) was performed for quantification of PGE2 in cell culture supernatants per the manufacturer’s recommendations. The plate was read by using a Cytation3 imaging plate reader (BioTek, VT, United States).

For meloxicam response regarding total cell number and metabolic activity, cells were seeded in 24-well plates 4*10⁴ cells/well and in 96-well plates 7,500 cells/well. The next day, medium was exchanged and cells were exposed to 1μM, which resembles the therapeutic steady state plasma concentration in dogs [66 (link)], and 10 μM meloxicam in quadruplets. Controls were incubated with 0.13% (v/v) DMSO. After 72 h of exposure, the MTS assay procedure was conducted as described for doxorubicin and carboplatin inhibition and cells were counted as described for doubling times.
The functionality of COX-2 enzyme was ascertained by production of prostaglandin E2 (PGE2). Each cell line was seeded in a density of 7.5*10⁴ cells/well in 12-well plates. The next day, medium was changed and cells were treated in duplicates with 1 and 10 μM meloxicam. Controls were incubated with 0.13% (v/v) DMSO. After 24 h of exposure, medium was collected, centrifuged for 10 min at 160 g for removal of cells and debris and stored at -80 °C. PGE2 concentrations in medium samples were measured in duplicates using a competitive ELISA (Prostaglandin E2 ELISA monoclonal, Cayman Chemical, Ann Arbor, USA) according to manufacturer’s protocol.