Procedures have been accepted by the Institutional Ani mal Care and Use Committee with the University of Colorado. Isolation of lung protein exudates and alveolar macrophages Primary alveolar macrophages and lung protein exudates were isolated by bronchoalveolar lavage from male A J mice 24 32 wks following a single i. p. injection of ten mg g ethyl carbamate or 0. 9% NaCl vehicle handle, as previously described, This dose of urethane induces several lung tumors inside a J mice, which are primarily adenomas at 24 wks and progress to AC from 24 42 wks. BAL macrophages from manage animals are considered na ve, although macrophages isolated from lung tumor bearing mice are tumor educated, Generation of JF32 cells from principal lung tumor isolates Thirty two wks following urethane injection, male A J lung tumors were resected from the lung underneath a dissecting microscope. Fifty mg of tumor tissue was placed onto a sterile Pyrex petri dish, finely chopped in 0.
two mL PBS which has a sterile razor, and also the resulting suspension extra to a Krebs Ringer buffered answer containing ten U mL Dispase 10 U mL collagenase I, The tumor suspension was digested with agitation for 60 min. at 37 C, soon after which digestion was terminated by adding an equal volume of 20 mM EDTA. The tumor suspension was then passed twice through a 20 ga syringe needle, and natural product libraries filtered to make just one cell suspension of tumor cells, as described for your isolation of principal Clara cells, These tumor cells have been washed three times in 10% FBS MEM a, collected by centrifugation, and their viability determined by trypan blue exclusion making use of a hemocytometer. The main tumor isolates selleckchem have been 90% viable by this process. Twenty thousand cells per well were plated in 1% FBS MEM a on Matrigel coated six very well plates, The primary tumor cell cultures have been maintained for 4 weeks, and MEM a media containing 1% FBS transformed after weekly.
For three weeks, there was tiny morphological modify in colony size or amount, and after that actively proliferating colonies were observed. Two adherent colonies were eliminated, designated JF32a and JF32b, plated onto standard 100 mm tis sue culture handled plates, and cultured as described beneath. Exon two in the Kras gene was sequenced as pre viously described, and Q61R Kras mutations detected in the two JF32a and b, consistent with our pre viously published report of Kras mutation incidence in urethane induced mouse lung tumors, Cell culture The non tumorigenic, mouse type II pneumocyte derived epithelial cell line was applied to represent non transformed lung epithelium in vitro.