I have found a few questions here for this issue but I am not sure about the current admitted idea.
I want to make a drug treatment for establishing a resistant line and I will use a nucleoside analog. So, should I make my cell population synchronic before treatment? Of course, I will make cell viability and cytotoxicity tests on them.
Some researchers said the starvation or serum deprivation is not proper for making the cells synchronic, while some assumed this method is working. If I try this manner, how can I sure about its trustworthiness? And if it is working, how much durable is it? E.g; for the next passage after the synchronization, should I do it again and again?
Thank you!
Hi Gülşah Torkay ,
Have you considered double thymidine block? If you look at the principles of double thymidine block (why we do blocking 2 times not once), maybe double cell starvation will be more efficient.
You can try to look at the stage of the cell cycle by flow cytometry (simple PI staining).
I have never checked the cells after subculturing to see if they are still synchronised or not (but I guess they will be for 2-3 passages). Just to be 100% sure you can set up a small experiment and do a PI flow cytometry after few passages.
Good luck.
cell cycle analysis is the option to go for. While many drugs induce cell cycle arrest at the g1 phase, others induce arrest at g2/m.
Dear Arman Javadi thank you for your answer. I want to ask one more thing; if I make double starvation, could it be something like subpopulation selectiveness? I mean will the remaining cells could be more resistant amid the population?
Hi Gülşah Torkay,
When you do the double starvation, majority of your cell population will be synchronized and the rest will be at other stages of cell cycle. So yes you will have some sub populations that at different stages (but their numbers will be very low).
If that worries you there is a technique with double thymidine block and nocodazole treatment. Once you treat the cells with Nocodazole cells will be synchronized at G2/M phase . Since mitotic cells are less adherent you can wash them and use it for your experiment (you will have 100% mitotic cells). After that you can grow the cells in normal media and perform a time assay to see how long it will take for cells to pass to other stages of cell cycle. However some cells do not tolerate nocadazole.
I agree with Arman, you can do the double thymidine block. As for starvation, it may not work on some cell lines, for example, Hela cells. As for double thymidine block, it can synchronize most but not all cells, which can be further analyzed by flow cytometry (by quantification of the DNA contents). Synchronization only strickly lasts for one cell cycle. As time goes by, more cells would be in different stages. I have read these somewhere, but I didn't further check them. Wish you all the best.
Dear Hamadi Madhi cell cycle analysis is an option yes, but I am trying to understand whether a synchronization is necessary or not. Because I do not have thymidine in my lab at the moment and if the serum deprivation works, I can try it. Maybe I can make a flow cytometer after it.
Dear Arman Javadi , I have understood the process but I am trying to figure out if these remaining and synchronised cells will more resistant than the killed ones or not. Because I want to establish a resistant cell line from a sensitive one. I do not want to sort a sub-population from the normal population during the double starvation. I have thought that maybe the starvation or serum deprivation could change my cell line profile. Hope that I did explained my problem well. Thank you very much again.
Hi Gülşah Torkay ,
Cell starvation does change the cell profile (specially if you are using cancer cells), the reference below is one example of it: Article Serum Starvation Induces DRAM Expression in Liver Cancer Cel...
So yes the selected cells using your method will be a sub-population that are more resistant to cell starvation.
Best,
Hello Gülşah,
To your question:" So, should I make my cell population synchronic before treatment? ", the short answer is: No.
You can do this but I don't think it's necessary, or even very useful. Your cells will not immediately be resistant to the drug and you don't want them to die too much, so you are going to start with low, sub lethal doses. The cells not in S phase will eventually "run" into it and then incorporate your analogue.
Another tip: Work with at least two dishes/flasks at the same time, one with a higher one with a lower concentration of the drug. If you then, in the course of your resistance trial, "loose" the cells with the higher concentration (i.e. to massive cell death or senescence) You will have the backup with the lower conc. to go ahead with.
Hope that helps.
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