LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
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Synopsis<br />
5.2 Measurement of γ-H2AX by flow cytometry after γ-radiation exposure: 0.5x10 6<br />
cells were irradiated with 2 Gy and 5 Gy γ-radiation in two sets. Unirradiated cell<br />
treated in the same way served as control. Following 30 min incubation from one set<br />
and 4 h incubation from the other set, cells were collected and washed with PBS and<br />
fixed with paraformaldehyde. Permeabilization was performed by addition of 0.3%<br />
TritonX-100 in FACS buffer (2% FBS and 0.02% sodium azide in 1X PBS) followed<br />
by blocking in 3% BSA in FACS buffer. Cells were incubated with γ-H2AX primary<br />
antibody at 1:250 dilution overnight at 4 o C and next day the cells were incubated with<br />
Alexa-488 secondary antibody at 1:300 dilution. Cells were further diluted with FACS<br />
buffer and acquired by flow cytometry. Data analysis was done using CellQuest Pro<br />
software.<br />
6. Effect of genotoxic exposure on cell cycle profile: In order to assess the effect of<br />
genotoxic exposure on cell cycle profile the following experiments were done.<br />
6.1 Effect of γ-radiation on cell cycle profile: 1x10 6 cells were irradiated with 5 Gy<br />
and 10 Gy radiation dose and allowed to grow for 10 h. Cells were collected, fixed and<br />
stained as described in 4.2. Cell cycle phase distribution was obtained after analysing<br />
data using ModFit LT V 2.0 software. The differences in G2 cell percentages between<br />
treated cells and untreated cells were recorded as the G2 cell cycle delay percentage.<br />
6.2 Effect of BPDE exposure on cell cycle profile: 1x10 6 cells were treated with 5 µM<br />
and 8 µM BPDE dissolved in Tetrahydrofuran (THF) and incubated for 6 h. Untreated<br />
cells and cells treated with THF alone were taken as controls. After 6 h half of the cells<br />
were collected and fixed with 70% alcohol. Rest half were further allowed to grow for<br />
18 h, after removing BPDE containing medium and adding fresh complete medium, so<br />
as to complete a 24 h cell cycle. After the 24 h rest half of the cells were collected and<br />
fixed with 70% alcohol. The cells for both BPDE concentration and both time points<br />
were stained as described in 4.2. Cell cycle phase distribution was obtained after<br />
analysing data using ModFit LT V 2.0 software.<br />
7. Measurement of percent cell death after genotoxic exposure: LCLs were treated<br />
with γ-radiation and BPDE to assess difference in percent cell death between MPN<br />
patient and control groups.<br />
7