I am doing a reprogramming process to generate iPS using Doxycycline inducible OSKM polycistronic lentiviral cassette. But just 24 hours after the first treatment all the Dox treated plates (both cancer and non cancer cells) start to die and deattach from the surface while the non treated cells are normal and viable, so I can not keep the process. Is someone who experienced the same situation? Or does someone know why this happens to my cells?
It sounds like something in your treatment is somehow killing them. I would've guessed that the lentiviral particles were the source of the toxicity (in which case, you could incubate them for less time), but your transduced, non-treated control cells are fine.
I suppose I can speculate. When you prepare your doxycycline dilution, are you filter-sterilizing it before treatment?
I've attached the Mouse STEMCCA Dox-Inducible Polycistronic (OKSM) Lentivirus Reprogramming Kit protocol from Millipore.
I have not been working with this system yet, so I also just can speculate about the boundary conditions:
Which type of cells do you want to induce and how fast do they proliferate? What is the confluence at the beginning of your experiment?
Do you expect a toxic effect of doxycyclin at the given concentration? I mean, is the treatment harsh enough to kill a portion of the population? You might want to quantify that in a preliminary experiment.
If yes, the culture conditions of your cells are important: If they become confluent during the process and a portion of the cells die, you might observe detachment of the whole cell monolayer, including the cells which might survive the process. In this case, decreasing the cell density might help to recover the successfully reprogrammed cells.
Thanks Joseph and Matthias for the helpful answers.
I am not wondering that the effect would be a result of Doxycycline toxicity because this concentration is in use in our lab for the same cells.
My target cells are Ras oncogen knocked in glioblastoma cells which proliferate fast. But we are using MEF cells in parallel but the same thing happen for them as well by Dox treatment. The same thing happened also to different cell confluence cultures. It appears that the effect might be due to OSKM overexpression. Because Dox alone was not able to cause the same problem.
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