Exon two on the Kras gene was sequenced as pre viously described, and Q61R Kras mutations detected in each 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 kind II pneumocyte derived epithelial cell line was used to represent non transformed lung epithelium in vitro. To study the interactions of tumor cells with macrophages, 3 neoplastic mouse lung cell lines had been utilised, the newly generated JF32a cells, LM2, previously derived from a urethane induced lung tumor inside a J mice, and E9, a spontaneous transfor mant of E10 cells. Culture of all cell lines was previously described, JF32 cells had been maintained like the LM2 cell line.
To study the in vitro effects of immune mediators on epithelial cells, MH S macrophages, an alveolar macrophage cell line iso lated from a BALB c mouse, or main BAL macrophages had been made use of. All macrophages had been main tained in RPMI 1640 inhibitor PF-04217903 in accordance with ATCC recommendations for the MH S cell line. All cells had been cultured inside a humidified atmosphere of 5% CO2 in ambient air at 37 C, and routinely screened for Myco plasma contamination. Exactly where indicated, 2 50 ng mL recombinant mouse IGF 1 and or EGF were added to epithelial cultures. LM2 and JF32 cells have been suspended in 0. 5% low melting point agarose in MEM a media containing 0. 5% BSA, and plated at 1,000 cells effectively into 12 nicely plates using a pre coated base layer of 1% agar, and also a best layer of 0. 75% LMP agarose. After weekly, cells have been fed with 0. five mL MEM a 0. 5% BSA or macrophage conditioned media.
Soon after 5 6 wks of development, colony number was deter mined under 20? magnification having a bright knowing it field inverted microscope. Alternatively, neoplastic cells had been suspended in MEM a media containing 0. 5% BSA, and plated at 3,000 cells nicely onto ultra low attachment six effectively culture plates. Cells had been fed as soon as weekly with 1 mL MEM a 0. 5% BSA or macrophage conditioned media. Immediately after three wks, the contents of each effectively were removed with a pipette, and cells pelleted by 5 min. centri fugation at 600 ? g. Cells had been resuspended in 1. 5 mL Accutase, and incubated for 20 min. at 37 C to make a single cell suspension. Equal volumes of cell sus pension were added to 0. 4% Trypan blue solution, and live vs. dead cells ascertained employing a hemocytometer. Macrophage co culture and conditioned media Epithelial cell lines had been plated onto tissue culture trea ted plates. Macrophages have been plated onto 0. 4 um pore Transwell inserts to enable diffusible signals to exchange during co culture although preventing physical contact. Epithelial cells and macrophages have been plated separately in media containing 10% FBS and permitted to equilibrate more than evening.