LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
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Materials and Methods<br />
specific binding. Cells were further incubated with Alexa-546 secondary antibody at<br />
1:200 dilution in blocking buffer for 1 h at RT. Cells were washed for the second time<br />
with PBS and PBST thrice alternately to get rid of nonspecific antibody binding. Nuclei<br />
were counterstained with DAPI and to remove excess DAPI cells were washed twice<br />
with PBS. Coverslips were mounted on glass slide using Dabco as anti-quenching<br />
agent. Edges of the coverslip were sealed with nail paint. γ-H2AX was visualized as red<br />
foci on DAPI stained nuclei and acquired on confocal microscope.<br />
3.5.2 Measurement of γ-H2AX by flow cytometry after γ-radiation exposure:<br />
Materials:<br />
1. Cobalt-60 isotope source (<strong>Bhabha</strong>tron-II)<br />
2. 1% PFA<br />
3. FACS buffer (PBS containing 2% FBS and 0.02% Na-azide)<br />
4. 0.3% TritonX-100 in FACS buffer<br />
5. BSA<br />
6. Anti γ-H2AX primary antibody<br />
7. Anti mouse Alexa-488 secondary antibody<br />
Method:<br />
0.5x10 6 cells were seeded in 1ml complete DMEM in 35 mm culture plates<br />
and irradiated with 2 Gy and 5 Gy γ-radiation in two sets. Unirradiated cells treated in<br />
the same way served as control. Following 30 min incubation from one set and 4 h<br />
incubation from the other set, cells were collected and washed with FACS buffer and<br />
fixed with 1% PFA for 15 min at RT. Cells were washed with PBS and permeabilized<br />
by addition of 0.3% TritonX-100 in FACS buffer for 15 min at RT. Cells were again<br />
washed with FACS buffer followed by blocking in 3% BSA in FACS buffer for 1 h.<br />
Next the cells were collected by centrifuging at 1500 rpm/ 10 min/ RT and incubated<br />
with 50 μl γ-H2AX primary antibody at 1:250 dilution in blocking buffer overnight at 4<br />
o C. Next day the cells were washed with FACS buffer and incubated with 50 μl Alexa-<br />
488 secondary antibody at 1:300 dilution in blocking buffer. Cells were once again<br />
washed with FACS buffer to get rid of any unbound antibody and further diluted with<br />
200 μl FACS buffer and acquired on flow. Data analysis was done using CellQuest Pro<br />
software.<br />
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