Prostaglandin e metabolite eia kit

The Prostaglandin E Metabolite EIA Kit and the 13,14-dihydro-15-keto Prostaglandin F2α ELISA Kit (Cayman Chemical Company, Ann Arbor, MI, USA) were used to measure the levels of PGE₂ and PGF_2α metabolites in LsHPX1 KD copepodids and adult lice homogenates, according to supplier’s instructions with some modification as follows. For the PGE₂ analysis, around 200 copepodids were pooled or one female was homogenized in 75 µl ddH₂O using 1.4-mm zirconium oxide beads (Precellys 24; Thermo Fisher Scientific) and a TissueLyser LT (Qiagen) for 4 min at 50 Hz. Samples were centrifuged for 5 min at 1.5 g, and 55 µl of the supernatant was added to 16.5 µl carbonate buffer. After an overnight incubation on 37 °C, 22 µl of phosphate buffer and 16.5 µl EIA buffer were added, and 50 µl of the sample was added to two wells (two technical replicates). For the PGF_2α analysis, around 150 copepodids were pooled in 100 µl ddH₂O (N = 3) and homogenized as described above. Samples were centrifuged for 5 min at 1.5 g, and 50 µl of the supernatant was added to the ELISA wells. Two technical replicates were run for each of the three biological replicates.

Protein lysates (30 μg per sample) were separated by SDS-PAGE and transferred to PVDF membrane for immunoblotting. Anti-COX-2, anti-MUC1 TR (extracellular portion of MUC1), anti-MUC1 CT (cytoplasmic tail of MUC1), and anti-IL-10 antibodies (SantaCruz Biotechnology, Dallas, TX, USA) were used.
PGE₂ ELISAs were performed using the Prostaglandin E₂ EIA Kit-Monoclonal kit (Cayman Chemical; Ann Arbor, MI, USA) on TCCM and FCM. PGEM (13,14-dihydro 15-keto PGA₂) ELISAs were performed using the Prostaglandin E Metabolite EIA Kit (Cayman Chemical; Ann Arbor, MI, USA) on tumor lysates and serum from tumor-bearing mice. PGE₂ is metabolized rapidly in vivo and hence an accurate measurement of PGE₂ is not possible from in vivo samples, therefore the PGE₂ metabolite (PGEM) concentration is assessed in the serum and tumor lysate of tumor-bearing mice.

Urinary PGE-2 levels were determined using the Prostaglandin E Metabolite EIA kit (Cayman Chemical Co.).

Plasma IL-6, IL-1β and TNF-α cytokines were quantified by the ELISA method (Bioscience, San Diego, USA). Prostaglandin E₂ metabolites were quantified by competition ELISA method using the Prostaglandin E Metabolite EIA kit (Cayman Chemical Company, USA) according to the manufacturer’s instructions.

The serum PGE₂ levels were measured by enzyme immunoassay (EIA) using a prostaglandin E metabolite EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, aliquots of prepared standard solutions and sample sera were plated in 96-well plates from the PGE₂ EIA kit, and a PGE₂-acetylcholinesterase (AChE) conjugate (PGE Tracer) and PGE₂ monoclonal antibody solution were added and allowed to react at 4 °C for 24 h. Next, Ellman’s reagent, which contains an AChE substrate for color formation, was added to the wells and the optical density was measured at 412 nm (VersaMax ELISA Microplate Reader; Molecular Devices, Sunnyvale, CA, USA). The PGE₂ concentrations in the serum samples were determined using a four-parameter logistic equation (logit(B/B₀) = ln[B/B₀/(1–B/B₀)]) by comparisons with the absorbances of the PGE₂ standard solutions at several concentrations (8, 16, 31, 62, 125, 250, 500, and 1000 pg/mL).