Cold ischemia was simulated by replacing cell culture medium with cold Perfadex® solution, a clinically used solution for donor lung preservation, and stored in a 4ºC chamber for 6 h, 12 h, 18 h, 24 h. Clinically, donor lungs are inflated with 50% O2 during hypothermic preservation, thus, the chamber was filled with room air mixed with 50% O2. To simulate reperfusion, the cold Perfadex® solution was changed back to warm serum-containing culture medium and cultured at 37ºC for 2 h or 4 h.
Growth protocol
Normal human bronchial epithelial cell line (BEAS-2B) was obtained from ATCC (Manassas, VA), and cells were cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, GIBCO, Carlsbad, CA). Human pulmonary microvascular endothelial cell line (HPMEC) was obtained from Kirkpatrick’s research lab, and cultured in flasks or on plates precoated with 0.2% gelatin. The culture medium used was medium M199 supplemented with 20% FBS, Glutamax (2 mM), endothelial cell growth supplement from bovine neural tissue (ECGS 50 μg/ml, Sigma Aldrich), heparin (50 U/ml) and Penicillin/Streptomycin solution (100 U/100 μg/ml)
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified using RNeasy Mini Kit (Qiagen; Hilden, Germany). RNA quality was tested using Nanodrop spectrophotometer (VWR) and Bioanalyzer (Agilent; Santa Clara, CA).
Label
biotin
Label protocol
Affymetrix protocol
Hybridization protocol
Affymetrix protocol
Scan protocol
Affymetrix protocol
Data processing
R oligo package, annotations from brain array v19, RMA normalization
