I am considering cell-cycle-synchronising a slow-dividing cell line (say, 1 week doubling time). Has anyone had any relevant experience? In particular, any suggestions how to relate the more-or-less standard procedure of synchronising fast cells (e.g. HeLa, MCF-7) by a double thymidine block (16-24 hours on, 8 hours off, 16-24 hours on) to the doubling time?
I am sorry. The method of double thymidine block of slow or fast growing cell lines used in the 1970s. There are many published literature. We did not recommend due to that the double thymidine block would result in the dTTP levels increasing, and feedback inhibition. It would be to disturb the stability during the cell cycle. Now flow cytometer is a good way to cell-cycle-synchronizing.
Thanks, Ellen. You are, of course, correct about the advantages of flow cytometry, although the essential trypsinisation and single cell suspension are in themselves not inert, and re-plating sorted cells into a 2D culture loses much of the synchrony. In any case, flow-sorting is not an appropriate method for me as my 2T block is a control for estrogen deprivation in a 2D culture to distinguish between genuine estrogen effects and accompanying effects of cell cycle. I am not familiar with the old literature for 2T block, perhaps I am not using the right search terms. Could you perhaps recommend a key publication talking about slow cell lines?
Thanks,
Anna
Hi, Anna : There are some published papers which can be to help you. Good Luck.
Kozaki Y, Amoh T, Yamaguchi N, Muramatu M. Fluctuation of trypsin-like proteinase activity in the cell cycle of synchronized and growing HeLa cells, and the effect of GMCHA-OPhBut. Biochim Biophys Acta. 1989 Nov 20;1014(2):120-4.
Fedarko NS, Ishihara M, Conrad HE. Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan. J Cell Physiol. 1989 May;139(2):287-94.
Coffman FD, Haviland DL, Green LM, Ware CF.Cytotoxicity by tumor necrosis factor is linked with the cell cycle but does not require DNA synthesis. Growth Factors. 1989;1(4):357-64.
Select item 7042716
Tanuma S, Kanai Y. Poly(ADP-ribosyl)ation of chromosomal proteins in the HeLa S3 cell cycle. J Biol Chem. 1982 Jun 10;257(11):6565-70.
Zucker RM. Cell cycle dependency of tumor promoter on murine erythroleukemic cells.
Cytometry. 1981 May;1(6):373-6.
Tupper JT, Mills B, Zorgniotti F. Membrane transport in synchronized Ehrlich ascites tumor cells: uptake of amino acids by the A and L system during the cell cycle. J Cell Physiol. 1976 May;88(1):77-87.
Lee SH, Rozee KR. Variation of interferon production during the cell cycle. Appl Microbiol. 1970 Jul;20(1):11-5.
Hello Anna!
You can find (I hope) helpfull information about cell synchronization in my review
from 2004. Full text is on my ResearchGate page and in attach file.
Best regards
Rustem Uzbekov
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