2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

Quantification of PGD₂ and 15d-PGJ₂ in cell-free culture media was performed according to previous studies [30,31]. To detect PGD₂ and 15d-PGJ₂ in the culture medium, freshly prepared 15 μM PGD₂ was made by diluting a stock solution in cell-free DMEM containing 10% FBS and 1% penicillin–streptomycin, which was incubated in a humidified atmosphere containing 5% CO₂ at 37 °C. The culture medium was collected at 0, 0.5, 1, 2, 4, and 24 h, homogenized in methyl acetate, and centrifuged at 10,000× g for 10 min at 4 °C. The supernatant was evaporated, and then the residue was reconstituted in the assay buffer. PGD₂ was measured using the Prostaglandin D₂–MOX EIA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocol. The absorbance of samples was measured using the microplate reader SH-1000Lab (Corona Electric, Ibaraki, Japan) at a test wavelength of 412 nm. Similarly, 15d-PGJ₂ was measured according to the protocol of the 15-deoxy-Δ^12,14-PGJ₂ ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA). The absorbance of samples was measured using SH-1000Lab (Corona Electric, Ibaraki, Japan) at test and reference wavelengths of 405 and 570 nm, respectively.