Prostaglandin e2 elisa kit monoclonal

For cytokine quantification in culture supernatants, the following ELISA kits have been used: Human/mouse TGF beta 1 2nd Generation ELISA Ready-SET-Go (eBioscience), Mouse IL-6 DuoSet ELISA (R&D Systems), Mouse IL-10 DuoSet ELISA (R&D Systems), Mouse TNF alpha ELISA Ready-SET-Go (eBioscience), and Prostaglandin E₂ ELISA Kit-Monoclonal (Cayman Chemical) following the manufacturers’ instructions.

Samples of radiation‐treated cell‐conditioned media and control media were collected and frozen at −80 °C until analysis. Media samples from doxorubicin‐treated cells were collected by replacing media on cells treated with doxorubicin for 48 h with agent‐free growth media and culturing cells for a further 6 h at which time the media were collected and stored at −80 °C. Cells were lifted and counted for normalization of PGE2 to sample cell number. Prior to ELISA assay, the cultured media samples were thawed on ice and clarified by centrifugation at 15 000 g for 15′ at 4 °C. PGE2 levels were quantified using Prostaglandin E2 ELISA Kit – Monoclonal (Cayman, Ann Arbor, MI, USA) as per the manufacturer's protocol.

We assessed eicosanoid production by chondrocytes using the Prostaglandin E₂ ELISA Kit – Monoclonal, Leukotriene B₄ ELISA Kit and Thromboxane B₂ ELISA Kit, according to the manufacturer's recommendations (Cayman Chemical, Ann Arbor, MI, USA). The concentration of each eicosanoid was determined according to the manufacturer's recommendations.

Prostaglandin E₂ (PGE₂) production was evaluated using the Prostaglandin E₂ ELISA kit—monoclonal (Cayman Chemical, Ann Arbor, MI, USA), following the manufacturer instructions. Briefly, cells were seeded in 24-well plates at a density of 200,000 cells per well and allowed to attach for 24 h. Subsequently, cells were exposed to the different drugs for 24 h. Then, supernatants were transferred to clear 1.5 mL tubes and centrifuged at 8000 g for 10 min at 4 °C. Aliquots (50 μL) of each sample were added to the IgG coated plate, and 50 μL of the PGE₂ AChE added to each well, with the exception of the total activity and blank wells. Then, 50 μL prostaglandin E₂ monoclonal antibody was added to each well, except for the total activity, blank, and non-specific binding wells. Subsequently, the plate was incubated for 18 h at 4 °C. The plate was then washed five times and Ellman’s reagent (200 μL/well) added, followed by incubation of the plate in the dark for 1 h at room temperature. Finally, the color developed was measured in a Varioskan plate reader (Thermo Scientific, Waltham, MA, USA) at 410 nm.

PGE₂ was measured in liver by a commercial enzyme immunoassay test kit (Prostaglandin E₂ ELISA Kit-Monoclonal, Item No.514010, Cayman Chemical) according to manufacturer’s instructions. PGE₂ concentrations in the samples were calculated from their corresponding absorbance values via the standard curve. The PGE₂ level was expressed in pg/mg liver protein (protein determination was performed by Lowry protein assay [41 (link)]).