Prostaglandin e2 eia kit

Rat microglial cells were purified from forebrains and cultured as previously described [32 ]. Cells were seeded on poly-d-lysine-coated plaques at a density of 50,000 cells/cm² and maintained for 3 days in DMEM containing 5% horse serum. Then, the cells were incubated for 18 h with 0.5 μg/ml LPS, in the presence of different concentrations of VCE-004.8, and supernatants were collected. Supernatants were spun down at 2000 rpm for 10 min, 4 °C. PGE2 in microglia was analyzed and quantified by using the prostaglandin E2 EIA Kit (Cayman chemicals) following provider recommendations.

PGE2 concentrations was evaluated in cell culture supernatants obtained from B16/CTRL, B16/PTGS2Δ, and B16/SCR. Prostaglandin E2 EIA kit (Cayman Chemicals, Ann Arbor, MI)) was used according to the manufacturer’s instruction.

PGE2 concentrations was evaluated in cell culture supernatants obtained after 24 h of treatment with the extracts (10 µg/mL) and stimulation with LPS/IFN-γ. A prostaglandin E2 EIA kit (Cayman Chemicals, Ann Arbor, MI, USA) was used according to the manufacturer’s instruction.

The concentration of PGE₂ in the conditioned medium was determined using a prostaglandin E₂ EIA kit (Cayman Chemical Company, Ann Arbor, USA) according to the manufacturer's instructions. The concentration of PGF_2α was determined using the direct enzyme immunoassay (EIA) method as previously described by Uenoyama et al. [31 (link)] with modifications. The standard curve for PGE₂ ranged from 16.5 ng/mL to 1000 ng/mL. The intra- and interassay coefficients of variation (CV) were 5.9% and 7.6%, respectively. The standard curve for PGF_2α ranged from 0.19 ng/mL to 50 ng/mL and CV were 5.5% and 8.6%, respectively.

To obtain conditioned medium (CM) generated from hAMTCs, freshly isolated hAMTCs were cultured for 3 days in 24‐well plates (Corning, NY, USA) at a density of 3 × 10⁵ cells/well in 0.5 ml Roswell Park Memorial Institute (RPMI) complete medium, composed of RPMI 1640 medium (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma‐Aldrich), 2 mm l‐glutamine (Sigma‐Aldrich) and 100 U/ml penicillin and 100 mg/ml streptomycin (pen–strep, herein referred to as P/S; both from Sigma‐Aldrich).
To obtain CM without prostaglandins (CM – PG), hAMTCs were cultured as described above in the presence of 10 μm indomethacin (Sigma‐Aldrich), a cyclooxygenase inhibitor. PGE2 quantification in CM and CM – PG was obtained using a Prostaglandin E2 EIA Kit (Cayman Chemical Co., Ann Arbor, MI, USA), according to the manufacturer's instructions. Absorbance was measured at 405 nm using a microplate reader.
At the end of the culture period, CM and CM – PG were collected, centrifuged at 300 × g, filtered through a 0.8 μm sterile filter (Sartorius Stedim, Florence, Italy) and kept frozen at −80 °C until use.
To obtain results that were least influenced by single donor variability and more representative of soluble factors released by hAMTCs, 10 pools, each containing CM from three to six different cell preparations, were used.

Nuclear protein was extracted by NE-PER Nuclear Protein Extraction Kit (Thermo). NF-κB (p65) binding activity and PGE2 were detected by NF-κB (p65) Transcription Factor Assay Kit (10007889, Cayman chemical), Prostaglandin E2 EIA kit (514010, Cayman chemical), respectively, according to the manufacturers’ instructions.