LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

SYNOPSIS
2.6 Immunofluorescence staining
Cells were grown in cover slips which were kept in 35mm petri dishes and irradiated as
mentioned above. Cell layer were washed 4 h after irradiation in PBS and fixed for 20
min in 4% paraformaldehyde at room temperature. Afterwards, the cells were washed
twice in PBS. For immunofluorescence staining, cells were permeabilized for 3 min in
0.25% Triton X-100 in PBS, washed two times in PBS and blocked for 1 h with 5% BSA
in PBS. Antibodies were diluted (1:200) in 1% BSA in PBS. Cells were incubated with
primary antibodies for 1 h 30 min at room temperature, washed three times in PBS and
incubated with secondary antibodies for 1 h at room temperature. Finally, cells were
rinsed and mounted with ProLong Gold antifede with DAPI mounting media (Molecular
Probe, USA). Images were captured using Carl Zeiss confocal microscope. Acquisition
settings were optimized to obtain maximal signal in immunostained cells with minimal
background.
2.7 Image analysis using ImageJ software
The captured images were analyzed for relative quantification of phosphorylation using
ImageJ software [10]. A 25 x 25 pixel box was positioned over the fluorescent image of
each cell, and the average intensity within the selection (the sum of the intensities of all
the pixels in the selection divided by the number of pixels) was measured. At least 100
cells per experiment were analyzed from three independent experiments.
2.8 Western Blotting
For the samples that had to be probed with specific antibodies, the proteins were
separated by SDS-PAGE (10%) followed by transfer to nitrocellulose membrane
(Hybond ECL, Amersham Pharmacia Biotech), and probed with specific antibodies. The
membranes were then probed with horseradish peroxidase conjugated secondary antibody
against mouse/rabbit (Roche Molecular Biochemicals, Germany) and developed using
BM Chemiluminiscence Western Blotting Kit Mouse/Rabbit (Roche Molecular
Biochemicals, Germany). Densitometry was done using Shimadzu CS 9000 Dual
wavelength flying spot scanner. Statistical analysis was done using ANOVA.
2.9 Measurement of DNA damage
DNA damage in all the groups of EL-4 cells 2 h after exposure to different conditioned
medium, was measured by alkaline single cell gel electrophoresis (comet assay) [11]. In
brief, frosted microscope slides (Gold Coin, Mumbai, India) were covered with 200 µl of
1% normal melting agarose (NMA) in phosphate buffered saline (PBS) at 45 o C, covered
with a cover slip and kept at 4 o C for 10 min. After solidification a second layer of 200 µl
of 0.5% low melting agarose (LMA) containing approximately 105 cells at 37 o C was
layered over it. The cover slips were replaced immediately and the slides were kept at 4
o C. After solidification of the LMA, the cover-slips were removed and slides were placed
in the chilled lysing solution (2.5M NaCl, 100mM Na 2 -EDTA, 10mM Tris–HCl, pH 10,
and 1% DMSO, 1% Triton X100 and 1% sodium sarcosinate) for 1 h at 4 o C. The slides
were removed from the lysing solution and placed on a horizontal electrophoresis tank
filled with freshly prepared alkaline buffer (300mM NaOH, 1mM Na 2 -EDTA and 0.2%
DMSO, pH≥13.0) and were equilibrated in the above buffer for 20 min and
electrophoresis was carried out at 25V for 20 min. After electrophoresis the slides were
washed gently with 0.4M Tris–HCl buffer, pH 7.4, to remove the alkali, stained with 50
µl of propidium iodide (PI, 20 µg/ml) and visualized using a Carl Zeiss Fluorescent
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