LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 2
MATERIALS AND METHODS
2.10 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) [228]. 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 microscope (Axioskop). The
images (40–50 cells/slide) were captured with high-performance GANZ (model: ZCY11PH 4)
color video camera. The integral frame grabber used in this system (Cvfbo1p) is a PC based card
and it accepts color composite video output of the camera. The quantification of the DNA strand
breaks of the stored images was done using the CASP software by which %DNA in tail, tail
length, tail moment and Olive tail moment could be obtained directly [229].
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