I am investigating mtDNA damage using a doxycycline-inducible cell line. The idea is to study this process at different timepoints, however, it only works fine until more or less 24 h post induction. Usually I start the experiment when the plates are 60-80% confluent (for the timepoints - 2, 4, 8 and 24 h). To be able to study further timepoints (48 and 72 h) I inoculated the plates with half the amount of cells. After induction I added doxycycline (and inhibitors when relevant) every 24 h (for 48 and 72 h timepoints).
The results are very inconsistent and we commonly see is an confluence-dependent increase in the amount of protein of interest.
I am wondering what would be the best way to deal with experimental design. What is the best confluence to begin with for the time-course treatment, how to split the cells to receive consistent results for further time-points. Should the doxycycline and inhibitors be added? How long do they remain active?
Any suggestions in these matter would be of great value.
Since your experiment is a Toxicological analysis on cells, it is very important to determine and identify a suitable subculture protocol. MOreover, characterization of cells is a must. Once, if you understand the cell's behaviour, you must perform volume and concentration based titrations with the chemical of interest on various cell densities to determine appropriate cell seeding. Once seeding is established, different end point analysis must be performed at various confluence levels (observational & experimental) of that particular cell seeding.
Always note that, the visual microscopic observation (observational) of cell confluence may not match with the actual cell yield (experimental).
Once all these standardization is validated by obtaining reproducibility and reliability with triplicated experiments.. It is the time to start real experiment.