LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 2
MATERIALS AND METHODS
2.2 Cell Lines
Human lung adenocarcinoma A549 cells, human breast carcinoma MCF-7 cells, human
monocyte U937 cells and human intestinal cell line INT 407 cells were cultured in 75 cm 2 tissue
culture flasks (Falcon, USA) in in Dulbecco’s modified eagles medium (Sigma) supplemented
with 10% fetal calf serum (Sigma). Mouse lymphoma cell line, EL-4 and mouse macrophage
RAW 264.7 cells were grown in RPMI 1640 (Sigma, USA), supplemented with 10% fetal calf
serum (Sigma). Cells were kept at 37 0 C in humidified atmosphere with 5% CO 2 in Nu-Air water
jacketed CO 2 Incubator (USA).
2.3 Irradiation
2.3.1 Low LET ( 60 Co γ rays): A549, INT 407 and MCF-7 cells were exposed to different doses
of γ irradiation using Gamma Cell 220 irradiator (Atomic Energy of Canada Ltd), at a dose rate
of 3.5 Gy/min; the first set was irradiated with acute dose of 2 Gy or 10 Gy. The second set was
exposed to 5 fraction of 2 Gy each over a period of five days.
A549 cells and PMA stimulated U937 cells were exposed to 2 Gy γ-irradiation. The
medium from irradiated cells was transferred to unirradiated cells. EL-4 and RAW 264.7 cells
were resuspended in RPMI medium at a density of 1x10 6 cells/ml and exposed to 5 Gy γ
irradiation using Gamma Cell 220 irradiator (Atomic Energy of Canada Ltd), at a dose rate of
4.74 Gy/min. Medium from irradiated cells was transferred to unirradiated cells (1 h post
irradiation). Some group of RAW 264.7 cells were stimulated with 500 ng/ml of bacterial
Lipopolysaccharide (LPS, Sigma) for 3 h and then the medium was replaced before irradiation.
2.3.2 Proton beam irradiation: Proton beam irradiation was carried out using the Radiation
Biology beam line of in-house 4 MeV Folded Tandem Ion Accelerator (FOTIA) facility. The
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