Mice were euthanized using an overdose of ketamine (160 mg/kg) and xylazine (25 mg/kg) at 1 and 7 days from challenge with CuO-NP. Bronchoalveolar lavage (BAL) was then performed as described previously [72 (link)]. Briefly, BAL was performed through a 20-gauge angiocatheter (BD Pharmingen, San Diego, CA, USA), with the intratracheal instillation of 1 mL of phosphate-buffered saline (PBS) containing an antiprotease cocktail (MilliporeSigma, Burlington, MA, USA) and 5 mM EDTA given in 0.5 mL increments [69 (link),70 (link),73 (link)]. An aliquot was immediately separated to measure total leukocyte cell counts (cells/mL BALF) using a Coulter Cell and Particle Counter (Beckman Coulter, Miami, FL, USA). The remaining lavage fluid was centrifuged at 1200 rpm for 10 min and the cell-free supernatant was frozen at −80 °C for cytokine and protein analysis.
The amount of total protein in the BALF was assayed using the BCA Protein Assay Kit (Pierce, Rockford, IL) as per manufacturer’s instructions. Absorbance was read at 560 nm (MRX Microplate Reader, Dynatech Laboratories, Chantilly, VA, USA) and protein levels in mg/mL of BALF were calculated. The results are reported as fold change from untreated control.
The amount of total protein in the BALF was assayed using the BCA Protein Assay Kit (Pierce, Rockford, IL) as per manufacturer’s instructions. Absorbance was read at 560 nm (MRX Microplate Reader, Dynatech Laboratories, Chantilly, VA, USA) and protein levels in mg/mL of BALF were calculated. The results are reported as fold change from untreated control.