Complete rpmi 1640

Total bronchoalveolar lavage fluid (BALF) was collected by lavaging whole lung through the tracheostomy angiocatheter. Total cells from BALF were enumerated and differential cell counting was performed on modified Giemsa-stained cytospin preparations. Plasma was harvested by collecting whole blood into 10% 0.5M EDTA (Thermofisher scientific, Waltham MA) coated tubes. The right lung superior, middle, and inferior lobes were harvested and processed as follows. Lungs were cut into small pieces and incubated in digestion buffer (2mg/ml collagenase (#LS004177, Worthington), 0.04mg/ml DNAse (#10104159001, Sigma) 1, 20% FBS in HBSS) for 1 h at 37°C after which they were deaggregated by pressing through a 40 μM nylon mesh and centrifuged at 400 x g for 5 minutes at 4°C. Supernatants were discarded, and 1.5 mL of ACK (Thermofisher scientific, Waltham MA) was added and incubated for 3 min at room temperature for erythrocyte lysis. ACK was then neutralized with 7.5 mL of complete RPMI-1640 (Corning, NY), with 10% FBS and 1% Pen Strep, (Gibco, Waltham MA). The resulting leukocyte single cell solution was centrifuged and prepared for flow cytometry analysis, Luminex or ELISA.

BM cells from B6.TC mice were cultured with or without 1 mg/mL hAAT in complete RPMI 1640 (Corning Cellgro, Manassas, VA) containing 10% fetal bovine serum (FBS) (Thermo Scientific, USA), 2- Mercaptoethanol (Sigma-Aldrich, MO), 10 ng/mL GM-CSF, and 5 ng/mL IL-4 (PeproTech, NJ). On day 3, 50% of the culture medium was replaced with fresh medium containing the same supplements. On day 4, cells were stimulated by adding 10 μg/mL CpG-ODN 1826 (InvivoGen, San Diego, CA) for an additional 24 hr. All cells were collected at day 5 and analyzed by flow cytometry, and the supernatant was stored at −80°C for cytokine detection.