Pge2 elisa assay kit

The production of PGE₂ was measured by enzyme-linked immunosorbent assay (ELISA) using PGE₂ ELISA assay kit (Enzo Life Science, Farmingdale, NY, USA). Briefly, cells were treated as previously described. The culture supernatants were then collected, after which the optical densities were measured using a spectrophotometer (TECAN, Männedorf, Switzerland) at a wavelength of 405 nm.

To measure PGE₂, the cells were plated at a density of 1 × 10⁶ cells/well in six-well plates, co-treated with PMA for macrophage differentiation, and treated with metformin and/or mevalonate for 48 h. The levels of PGE₂ in the supernatants were determined using the PGE₂ ELISA assay kit (ADI-900-001; Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s protocol. Enzymatic reactions were measured by spectrophotometry at 405 nm.

Auranofin was purchased from Sigma-Aldrich (St. Louis, MO). A solution of Auranofin was prepared in dimethyl sulfoxide, stored as small aliquots at -20 °C, and then diluted as needed in cell culture medium. FBS and RPMI 1640 medium were purchased from HyClone (Logan, UT). The Neon transfection system was from Life Technologies (Carlsbad, CA). The bicinchoninic acid (BCA) protein assay kit and ECL kit were from Thermo Scientific (Waltham, MA). PGE2 ELISA assay kit was purchased from Enzo Life Science (Farmingdale, NY). Antibodies against tubulin (sc-8035) and goat anti-rabbit IgG-Texas Red (sc-2780), and Ultra Cruz™ mounting medium (sc-24941) were from Santa Cruz Biotechnology (Santa Cruz, CA). COX-2 antibody (A303-600A-M) was from Bethyl (Montgomery, TX). NF-κB (p65) (CSB-PA003436), p-p65 (S536) (CSB-PA000586), p-p65 (S311) (CSB-PA000719), and PKC-ζ (CSB-PA003805) antibodies were from Flarebio (College Park, MD). All other chemicals were of the highest purity or molecular biology grade and were obtained from commercial sources.