LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 2
MATERIALS AND METHODS
2.11 Measurement of NO production in EL-4 cells
Control unirradiated cells, cells exposed to radiation and the cells exposed to different
conditioned media as mentioned above were cultured for 24 h at 37 0 C in 5%CO 2 / 95% air. The
production of NO in the culture supernatants was measured by assaying NO - 2 using colorimetric
Griess reaction [230].
In brief, 100 µl of culture supernatants was incubated with an equal volume of Griess reagent
[1% sulfanilamide and 0.1% N-(1-naphthyl-ethylenediamine) in 2.5% phosphoric acid; Sigma,
USA] at room temperature for 10 min in a 96 well plate. The absorbance at 550 nm was
measured using a microplate reader (Fluostar Optima, Biotron Healthcare). Absorbance
measurements were converted to µ moles of NO 2
-
using a standard curve of NaNO 2.
2.12 Measurement of Apoptosis by DNA fragmentation
Apoptosis in control unirradiated cells, cells exposed to radiation and the cells exposed to
different conditioned media as mentioned above was measured by DNA fragmentation assay
[231]. After transfer of medium from irradiated cells, all the above mentioned groups of EL-4
cells were cultured for 24 h at 37 0 C in 5%CO 2 , 95% air. The cells were harvested and lysed in
lysis buffer (10 mM EDTA, 50 mM Tris–HCl, pH 8.0, 0.5% sodium lauryl sarcosine) containing
the 100 mg/ml proteinase K at 55 o C for 2 h. The DNA was extracted with phenol/chloroform and
precipitated with ethanol. The RNA was removed by RNAse A treatment and DNA was
electrophorased on agarose gel to visualize the fragmented DNA.
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