4.3.3. Carrageenan-Induced Pleurisy

The mice (*n* = 6) were orally pre-treated with the vehicle, NTK (10 mg/kg), or indomethacin (25 mg/kg) 1 h before receiving an intrathoracic injection of 1% carrageenan (challenge). Four hours later, the animals were anesthetized with ketamine (8 mg/kg) and xylazine (8 mg/kg) and euthanized by cervical dislocation. The pleural cavity was washed with 1 mL of saline containing 0.9% Ethylenediaminetetraacetic acid (EDTA, Sigma, New York, NY, USA). The pleural lavages were centrifuged (5000 rpm, 5 min, at room temperature), and supernatants were stored (at −80 ◦C) for further analysis. The precipitate was added with 1 mL of PBS, and leukocytes were counted under light microscopy after diluting the pleural lavage samples in Turk fluid (2% acetic acid) [27].

#### 4.3.4. Quantification of MPO and Total Proteins

Samples of the peritoneal lavage were centrifuged at 6000 RPM for 2 min. Albumin quantification in the supernatants was performed using a Labtest kit (Lagoa Santa, Brazil) and used as a parameter of protein extravasation due to increased vascular permeability. In this method, the absorbance of bromocresol green, which specifically binds albumin, is proportional to the concentration of the protein in the sample. The readings were performed using a spectrophotometer with absorbance adjusted between 600 and 640 nm. Leukocyte activity was determined by quantifying the MPO enzyme. To this end, a tube was added with 40 μL of the supernatant of the pleural lavage and 1960 μL of the o-dianisidine and H2O2 reagen<sup>t</sup> in PBS (pH = 6.0). The readings were performed using a spectrophotometer at 450 nm.
