Quantifying Inflammatory Mediators

Peritoneal lavage or in vitro supernatants were analyzed for TNFα, CXCL1/KC, and LTB₄. TNFα and CXCL1 ELISA were performed with Duo Set kit, according to manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA) and LTB₄ with enzyme immuno assay (EIA) kit (Cayman Chemical, Ann Arbor, MI, USA). For the investigation of the presence of intracellular LTB₄, we used the previously described Eicosacell protocol (31 (link)). Briefly, leukocytes were recovered from peritoneal cavities and immediately submitted to fixation and permeabilization with 0.5% 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (Sigma) in HBSS. After that, a common immunodetection protocol was performed using the following antibodies: the primary antibody anti-LTB4 (Cayman Chemical) or irrelevant IgG and the secondary antibody Alexa 488-labeled anti-rabbit IgG. Images were obtained using an Olympus BX51 fluorescence microscope and equipped with a Plan Apo ×100 objective and a DP72 camera (Olympus Optical, Japan) in conjunction with Cell^F Imaging Software (Olympus Life Science Europe, Germany).