In brief, tissues were harvested directly into a volume of staining solution sufficient to cover the tissue and placed under a house vacuum for 10 min. Staining was carried out at 37°C in the dark for 48 hr.
At 4 hr post exposure, 20 µl of the cell suspension were mixed with equal volume of staining solution (100 µg/ml of propidium iodide and 0.5 µg/ml of acridine orange in phosphate-buffered saline).
The volume of interest was calculated by quantification of the volume of staining referenced to the surface area of basal lamina surveyed (cubic micrometre per square micrometre).
This volume of staining solution remained on the surface through surface tension during a 60-min staining period at room temperature in the dark.
This method can be performed without a microscope, but requires a large volume of staining reagents, and the sensitivity is low.
Cells were then counterstained with 0.5 μg/ml PI on ice for 5 min, and an equal volume of staining buffer was added.
After overnight fixation at −20°C, DNA was stained in an appropriate volume of staining solution containing 0.25 mg/ml propidium iodide, 0.05 mg/ml RNase and 0.1% Triton X-100 in citrate buffer, pH 7.8.
Inter-loci distances were normalized to nuclear diameter: first, the nuclear volume was calculated from the total volume of DAPI stained DNA; a spherical approximation was then used to calculate the diameter of the nucleus from this volume.
The latter process involved the addition of 30 μL of 7% paraformaldehyde to the plate, followed by 1-hour incubation at room temperature before aspiration of 60 μL of well volume and addition of stain.
To compensate for subtle differences in sample loading, gel staining, and destaining, the volume of each spot (i.e. spot abundance) was normalized as a relative volume.
After 24 hrs of radiation, the cells were suspended in equal volume of trypan blue stain (0.4% w/v) and incubated for 5 min. Then, we counted the cells using the Countess Automated Cell Counter (Invitrogen, CA).
