Patients with pre operative chemo ceritinib novartis therapy were excluded from the study. Surgical specimens were dissected into small fragments using a razor blade and fragments were incubated in 35 mm Petri dishes in 2 ml of DMEM F 12 growth medium containing 10% fetal calf serum, 2 mM L glutamine, 100 U ml penicillin, and 100 ug ml streptomycin. The study protocol was approved by the ethics review board of the Medical University of Graz. Signed informed consent was obtained from all patients prior to surgery. Cells The human NSCLC cell lines A549 and A427 were pur chased from Cell Lines Service and cultured in DMEM F 12 medium containing the sup plements described above. The human NSCLC cell lines NCI H23, NCI H358, NCI H1299, and NCI H441 were purchased from American Type Culture Collection and cultured in RPMI, supple mented with 10% fetal calf serum and antibiotics.
Carcinoma associated fibroblasts were Inhibitors,Modulators,Libraries isolated from three fresh NSCLC samples as described and cultured in DMEM supplemented with 10% fetal calf serum and antibiotics. CAFs were identified to be positive for vimentin and negative for cytokera tin using immunofluorescence. The purity of the cells was 97 99%. Human lung fibroblasts were cultured from donor lungs that could not be used for transplant ation as previously described. Hypoxic culture Fragments were cultured for three days at 37 C in ambi ent oxygen or 1% oxygen in the automated Xvivo System G300CL. NSCLC cells or fibroblasts were plated into cell culture flasks at 13,000 cm2 and let attach, thereafter cells were cultured for three days in ambient oxygen or 1% oxygen as de scribed above.
Exposure to oxygen Inhibitors,Modulators,Libraries was controlled through out the experiments in the hypoxic workstation. MTT assay The MTT assay was per formed on cultured fragments according to the manu facturers instructions. Briefly fragments were incubated in the MTT substrate solution for one hour and forma zan was dissolved in isopropanol. Inhibitors,Modulators,Libraries After dissolving the formazan 100 uL Inhibitors,Modulators,Libraries of sample was analyzed on a colorimet ric microplate reader at 570 nm. A549 cells were used as a positive control. Pimonidazole assay The assay was performed essentially according to the manufacturers instructions. Fragments were incubated for one or three days in hypoxia or normoxia. Thereafter fragments were treated with 100 uM pimonidazole HCl in hypoxia in the closed Xvivo Inhibitors,Modulators,Libraries hypoxic working chamber or in normoxia and incubated for one hour, fixed and paraffin embedded.
Bound pimonidazole was visualized using mouse monoclonal pimonidazole antibody. RNA extraction and cDNA synthesis Total RNA was extracted using the Qiagen RNeasy Mini kit and DNase digestion according to the manufacturers instructions. RNA integrity was assessed using the Agilent 2100 Bioa nalyzer and the Agilent therefore RNA 6000 Nano Kit.