I've recently thawed a TM4 Sertoli cell line, which I'd like to stimulate with a specific substance, but before I do that I want to synchronize it by using a specific Hunger medium.
I've read papers where they put the cells in serum-free DMEM/F-12 medium for 24h prior to the stimulation. I was thinking of adding 0,5% of FBS to DMEM/F-12 medium and incubating the cells for 24h instead of using totally serum-free medium.
What would be your suggestion for synchronizing these cells?
Note: For the culture of the cells I've been using the aforementioned medium with the addition of glutamine and 7,5% FBS.
Thanks in advance!
Dear Dimitris,
What do you want to synchronize in your cells? If you want to synchronize the DNA replication (cell cycle), starvation can be used, but also growing to plateau and the subculturing, eventually using conditioned medium (ie from a early log phase culture). An other approach would be via DNA synthesis inhibitors. I have obtained really good S-phase arrest with aphidicolin. It is expensive, but you only need very low concentrations to completely inhibit DNA sysnthesis at the beginning of S-phase . With plant cells, I routinely obtained >80% of synchronous passage of cells through S-phase after washing the cells free from Aphidicolin and resuspend them in conditioned medium.
I hope this is of help for you.
Greetings, Harrie
Better suggestion: don't use TM4. It may have some Sertoli cell characteristics, but it's incredibly abnormal.
http://www.karger.com/Article/FullText/48786 (pay per view)
http://www.spectral-imaging.com/attachments/250_Cytogenetic%20characterization%20of%20the%20TM4%20mouse%20Sertoli%20cell%20line.%20I.%20Conventional%20banding%20techniques,%20FISH%20and%20SKY.pdf (full text)
Tetraploid or more, with stable marker chromosomes and unstable karyotype between cells. I honestly wouldn't trust this to be representative of anything but itself.
Thank you both for your answers!
Harrie I want to synchronize the cells to the same phase of the cell cycle, so I'm guessing starvation alone should do the trick before I stimulate the cells.
Peter, I am simultaneously culturing the rat Sertoli cell line 93RS2, with which I also intend to perform the same stimulation with DHEAS. I use this specific cell line because of the fact that they carry an androgen and progesterone receptor, through which DHEAS potentially exerts its action. I just want to clarify the signalling pathway involved in DHEAS stimulation, which should probably remain the same regardless of the abnormalities in the karyotype of this cell line.
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