Gelcount colony analyzer

BH3 profiling and apoptosis (assessed by measuring the extent of phosphatidylserine (PS) externalization) were performed as described previously^{16 (link)}. For colony formation assays, a defined number of cells were exposed to different drugs and/or X-ray irradiation using a CellRad X-ray irradiator (Faxitron Bioptics) and incubated for 7–14 days. Cell colonies were fixed using 1:7 vol/vol acetic acid/methanol for 5 min and stained with 0.5% crystal violet (in 20% methanol) for 2 h, then counted on a GelCount colony analyzer (Oxford Optronix). Plating efficiency (PE) for the untreated controls and surviving fraction (SF) were determined using the formulae: $\begin{array}{c}\mathrm{PE}=\left[\mathrm{n}o.\mathrm{o}f\mathrm{c}olonies\mathrm{f}ormed\mathrm{∕}\mathrm{n}o.\mathrm{o}f\mathrm{c}ells\mathrm{s}eeded\right]\mathrm{and}\hfill \\ \mathrm{SF}=\left[\mathrm{n}o.\mathrm{o}f\mathrm{c}olonies\mathrm{f}ormed\mathrm{∕}\left(\mathrm{n}o.\mathrm{c}ells\mathrm{s}eeded\times \mathrm{P}E\right)\right]\times 100.\hfill \end{array}$

The indicated amounts of cells per well were plated on six wells or p60 plates (Fig. 4B only), let to adhere overnight and then treated as described. After that, the culture media were replaced every other day until the endpoint. Cells were then washed with PBS, fixed, and stained in a fixing/staining solution (0.05% crystal violet, 1% formaldehyde, 1% methanol in PBS) and washed abundantly. Images were acquired using GelCount colony analyzer (Oxford Optronix, Oxford, UK).