Quantifying PGE2 Levels in Nevus Samples

PGE₂ content in plasma samples was determined by ELISA using a PGE₂ assay kit (KGE004B, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions as described previously [22 (link)]. For nevus samples, frozen tissue was cut into sub-millimeter pieces and then homogenized in buffer (RD-556) supplied in the assay kit, using a disposable pestle (Kimble 749521-0590) obtained from Sigma-Aldrich, St. Louis, MO) on ice. After homogenization, nevus tissue lysates were sonicated briefly and then microfuged at 10,000 rpm for 10 min at 4 °C. After centrifugation, supernatants were collected, and protein concentrations were determined using a BCA protein detection kit (Thermo Fisher Scientific, Waltham, MA, USA). PGE₂ values were normalized to protein content for each sample.