‘Viability’ is usually measured with the trypan blue exclusion test. This is actually not a test of viability, but of membrane integrity. The membrane can be made leaky simply by scraping cells off the culture surface, but it soon gets repaired. In an experiment with HeLa cells, harvested by scraping or by trypsinisation, the scraped cells showed more than 80% trypan blue positive, whereas the trypsinised cells were 90% negative. But both gave the same low background damage scores.
Furthermore, the scraped and trypsinised cells were replated in fresh medium and cultured for 24 h. After that time, the number of attached, living cells was the same, whether they had been trypsinised or scraped. Really ‘unhappy’ cells, such as cells grown under sub-optimal conditions, will show high background levels of breaks. So – if in an experiment untreated, control cells give a low background level of DNA breaks, that is a good test of viability. Don’t rely on trypan blue.
If the density of comets in the gel is less than expected from the number of cells inoculated, this may be because there were dead cells, which disappear as the heavily fragmented DNA disperses.
"FAQ by Andrew Collins, Gunnar Brunborg and Jonas Nygren, 2006, NewGeneris FP7-project"