Ltb4 elisa

Resuspended neutrophils (35 μL) were made up to 70 μL with Dulbecco’s PBS containing methanolic cerumen extract (final concentration 1-500 μg/mL). Cell suspensions were incubated at 37 °C in a 5% CO₂ incubator for 20 min, then treated with 2 μM ionomycin for a further 5 min (final reaction volume 80 μL) to stimulate LTB₄ production [23 ]. Samples were centrifuged at 40×g for 6 min at 22 °C, and LTB₄ concentration was determined spectrophotometrically at 405 nm using 50 μL aliquots of the supernatant in a LTB₄ ELISA, according to manufacturer’s instructions (Cayman Chemical Company; Ann Arbor, USA). Background absorbance was measured in the absence of ionomycin and subtracted from all readings. Solvent (1% DMSO) and untreated controls were included in each assay, and were without effect (not shown).

Leukotriene B₄ (LTB₄), Prostaglandin E₂ (PGE₂) and Thromboxane B₂ (TXB₂) were quantified by the LTB₄ ELISA, PGE₂ Monoclonal ELISA and TXB₂ ELISA kits, respectively, all from Cayman Chemical (Ann Arbor, MI, USA).