LIFE09200604007 Tabish - Homi Bhabha National Institute

Synopsis
7.1 Percent cell death after γ-radiation exposure: 0.5x10 6 cells irradiated with 5 Gy
and 10 Gy γ-radiation and were allowed to grow for 48 h. Unirradiated cells treated in
the same way were taken as a control. To quantitate percent cell death cells were
stained with Annexin-V-FLUOS antibody and Propidium iodide (PI) where Annexin-
V-FLUOS stains apoptotic cells and PI stains the necrotic cells. Cells were incubated
with Annexin-V-FLUOS antibody at 1:100 dilution and 1 µg PI made in incubation
buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl 2 ) and analyze by
flow cytometry. Data analysis was done using CellQuest Pro software.
7.2 Percent cell death after BPDE exposure: 0.5x10 6 cells were treated with 5 µM and
8 µM BPDE dissolved in tetrahydrofuran (THF) and incubated for 6 h. Untreated cells
and cells treated with THF alone were taken as controls. After 6 h cells were collected
and washed with PBS. To quantitate percent cell death Annexin-V- PI staining was
performed as described in 7.1.
8. Genotyping of genes: Genotyping of genes was done using standard techniques as
described below.
8.1 DNA isolation: RBCs were lysed using sucrose lysis buffer (0.32 M sucrose, 10
mM Tris pH 7.5, 5 mM MgCl 2, 1% Triton X-100). Lysis buffer was added to blood in
the ratio of 10:1 and centrifuged to get the nucleated blood cell pellet. Genomic DNA
from the pellet was extracted by the lysis of cell membrane with STE buffer (0.1 M
NaCl, 0.05 M Tris pH 7.5, 1 mM EDTA pH 8.0, 1% SDS) and proteinase K digestion
over night at 37 °C. DNA was isolated by following standard phenol-chloroform
extraction method and an aliquot was run on 0.8% agarose gel to check DNA integrity,
quality and yield. The samples were stored at 0 °C till further use.
8.2 Polymerase chain reaction: PCR reactions were carried using gene specific
primers in sterile thin walled microfuge tubes. The reactions were carried out in 25 μl
volumes containing 100 ng of DNA template where reaction mixture without DNA
served as negative control and the product was analysed by agarose gel electrophoresis.
The remaining product was used for either restriction fragment length polymorphism or
for SNaPshot reaction.
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