Prostaglandin f2α eia kit

Supernatants of the co-culture of endometrial epithelial cells with isolated lactobacilli and L. vaginalis were analysed for concentrations of IL6 and IL8 by ELISA, as well as of prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂) by EIA. Concentrations were measured after 24 and 48 h of co-culture using commercially available kits according to the manufacturer’s instructions [Bovine IL-6 Screening Set (Thermo Scientific); Human CXCL8/IL-8 DuoSet (R&D Systems, Wiesbaden, Germany); Prostaglandin F_2α EIA Kit (Cayman Chemical, Ann Arbor, USA); Prostaglandin E₂ EIA Kit (Cayman Chemical)]. Dilutions of the samples within the detection range were used for each ELISA or EIA. Cross-reaction of the antibody pairs of Human CXCL8/IL-8 DuoSet to bovine IL8 has previously been shown [36 (link)]. Cell culture supernatants were centrifuged twice (400 g and 16,200 g for 5 min, respectively) after harvesting to remove epithelial cells and bacteria. Supernatants were stored at -80°C until measurement. Each experiment was conducted in duplicate using endometrial epithelial cells isolated from three different animals. Concentrations of pro-inflammatory factors in cell culture supernatants were estimated using a microtitre reader (iMark Bio Rad, Bio-Rad Laboratories, Munich, Germany) by comparing with standard curves.

Apical and basolateral supernatants were analyzed for PGE₂ and PGF_2α concentration using an ELISA method following supplier recommendations (Prostaglandin F_2α EIA Kit, 516,011; Prostaglandin E₂ EIA Kit, 514,010; Cayman). The supernatants were diluted in the provided buffer as appropriate. Briefly, 50 µL of diluted sample were added to each experimental well, followed by 50 µL of PGF_2α or PGE₂ Ache tracer and 50 µL of PGF_2α or PGE₂ EIA antiserum. Subsequently, ELISA plates were incubated for 18 h at 4 °C. Plates were washed five times with the provided wash buffer, and 200 µL of Ellman’s reagent were added to each well. Finally, the plates were covered with a plastic film and allowed to develop in the dark. The absorbance was measured at 450 nm in a microplate reader. Positive and negative sera controls were added to each microtiter plate for normalization. Controls and sera samples were analyzed in duplicate. Each tissue culture supernatant sample was assayed in duplicate. The mean of the duplicate values was used for statistical analysis. The concentration of PGE₂ and PGF_2α in each sample was interpolated from the standard curve. The intra- and inter- assay coefficient of variations for prostaglandins (PGs) analysis were: PGF_2α—9.1% and 9.2% and PGE₂ -3.7% and 9.7% respectively.