Detectx prostaglandin e2 high sensitivity immunoassay kit

To evaluate reactive oxygen species (ROS) and nitric oxide (NO) levels, U937 cells were differentiated to macrophages with PMA (U937/PMA) and triggered by LPS in the presence or not of P.l 1 : 10 or P.l 1 : 100. Following 24 hours, ROS and NO concentrations were measured by using 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA, Thermo Fisher Scientific, San Jose, CA, USA) and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM Diacetate, Thermo Fisher Scientific), respectively. Cells were incubated with DCF-DA or DAF-FM Diacetate for 30 minutes in the dark, and the fluorescence was recorded using a 96-well plate reader (GloMax, Promega). The levels of prostaglandin E₂ (PGE₂) were quantified in the cell culture supernatant after 48 hours of exposure to LPS by using DetectX® Prostaglandin E2 High Sensitivity Immunoassay Kit (Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer's protocol.

To evaluate ROS and NO^· levels, CD14⁺ monocytes were triggered by LPS in the presence or not of RWP 20 or 200 μg/mL. Where indicated, cells were treated also with 5 mM sodium malate (Sigma-Aldrich) or 500 μM NADPH (Sigma-Aldrich). Following 24 hours, ROS and NO^· concentrations were measured by using 6-Carboxy-2′,7′-Dichlorodihydrofluorescein Diacetate (DCF-DA, Thermo Fisher Scientific) and 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FM Diacetate, Thermo Fisher Scientific), respectively, as previously reported [33 (link)].
For PGE₂ quantification, cells were exposed to RWP 20 or 200 μg/mL for 1 hour and, where indicated, cotreated with 5 mM sodium acetate (Sigma-Aldrich); then inflammation was induced by LPS. At the end of 48 hours LPS treatment; PGE₂ was measured by using DetectX® Prostaglandin E2 High Sensitivity Immunoassay Kit (Arbor Assays, Ann Arbor, MI, USA) as previously described [33 (link)].