LIFE09200604007 Tabish - Homi Bhabha National Institute

Materials and Methods
Cobalt-60 isotope source (Bhabhatron-II) at RT so as to activate ATM in
phosphorylated form. Unirradiated cell line and lymphocytes treated in the same way
served as control. Following 30 min incubation cells were collected in 1.5 ml eppendorf
tubes. Cells were centrifuged at 1500 rpm to obtain a pellet and washed with PBS.
After PBS wash the pellet was resuspended in 200 μl PBS and carefully layered on
0.1% lysine coated coverslips. These cells were allowed to attach on coverslip for 5
min. After 5 min the remaining suspension was carefully pipetted back leaving the cells
attached on the coverslip. Cells were air dried to get rid of excess liquid and were then
fixed with 4% PFA for 15 min at RT. Cells were washed with PBS twice and
permeabilized by addition of 0.3% TritonX-100 in PBS for 15 min. Cells were again
washed with PBS twice followed by blocking with 3% BSA in PBST (0.1% Tween-20
in PBS) for 1 h at RT. Next the cells were incubated with pATM primary antibody at
1:150 dilution in blocking buffer overnight at 4 o C. Next day cells were washed with
PBS and PBST alternately thrice to get rid of any non specific binding. Cells were
further incubated with Alexa-546 secondary antibody at 1:200 dilution in blocking
buffer for 1 h at RT. Cells were washed for the second time with PBS and PBST thrice
alternately to get rid of nonspecific binding. Nuclei were counterstained with DAPI and
to remove excess DAPI cells were washed twice with PBS. Coverslips were mounted
on glass slide using Dabco as anti-quenching agent. Edges of the coverslip were sealed
with nail paint. pATM was visualized as red foci on DAPI stained nuclei and acquired
on confocal microscope.
3.4.5 Cell population doubling:
Materials:
1. 24 well plate
2. Trypan blue dye (Sigma)
3. Neubauer counting chamber
Method:
5x10 4 cells from LCLs were seeded for each time point in a 24 well plate with
1.5 ml of complete medium. Viable cell count was taken using Trypan blue dye
exclusion method at different time points including 0, 12, 24, 36, 48, 72 and 96 h as
described in section 4. For each time point four readings were taken.
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