C57BL/6J mice were infected intratracheally with 2.5 × 107 PFU Ad5mSPC-Cre (Dr. Anton Berns, Netherlands Cancer Institute ([email protected] )), after precipitation with 10mM CaCl2 for 20–60 minutes, and harvested on day 0, 1, 7, and 14 p.i.
Serum Isolation: Blood was collected retro orbitally, coagulated at room temperature for >30 minutes, and centrifuged at 1000xg for 10 minutes. Serum was diluted with sterile 1X phosphate buffered saline (PBS; ThermoFisher scientific cat. 10010023) per manufacturer instructions and frozen at −80 C. Lung homogenates: Whole lung tissue was removed following perfusion with 1X PBS, mechanically dissociated with scissors and homogenized with MP FastPrep 24–5g machine (MP Biomedicals cat. MP116913500) in 700 μL of RIPA 1x + HALT protease and phosphatase inhibitor (ThermoFisher Scientific cat. 89901, cat. 78440) on default mouse lung protocol. Samples were centrifuged at 1000xg for 10 minutes and protein was adjusted to 4 mg/mL after BCA protein assay (ThermoFisher Scientific cat. 23227). Samples were stored at −80 C until analysis. Serum and lung homogenates were analyzed by Eve Technologies Corporation using their “Mouse Cytokine Array/Chemokine Array” 44-Plex assay.
Serum Isolation: Blood was collected retro orbitally, coagulated at room temperature for >30 minutes, and centrifuged at 1000xg for 10 minutes. Serum was diluted with sterile 1X phosphate buffered saline (PBS; ThermoFisher scientific cat. 10010023) per manufacturer instructions and frozen at −80 C. Lung homogenates: Whole lung tissue was removed following perfusion with 1X PBS, mechanically dissociated with scissors and homogenized with MP FastPrep 24–5g machine (MP Biomedicals cat. MP116913500) in 700 μL of RIPA 1x + HALT protease and phosphatase inhibitor (ThermoFisher Scientific cat. 89901, cat. 78440) on default mouse lung protocol. Samples were centrifuged at 1000xg for 10 minutes and protein was adjusted to 4 mg/mL after BCA protein assay (ThermoFisher Scientific cat. 23227). Samples were stored at −80 C until analysis. Serum and lung homogenates were analyzed by Eve Technologies Corporation using their “Mouse Cytokine Array/Chemokine Array” 44-Plex assay.