Hello,
I want to stain MCF7 cells treated and untreated with beta- Gal.
My treatment lasts for 12 days, then I harvest the cells.
Does someone know how I could treat MCF7 cells to get a positive Control within max 10 days ? or even less ?
or any other way to treat a different cell line and get quickly Sen induction ?
I only know that irradiation of primary fibroblasts can induce Senescence and therefore beta-Gal positivity, but upon irradiation I would need to culture them 40 days and I don't have that time at the moment.
Thanks a lot in advance
24h treatment with 100 nM Doxorubicine, then keep them in fresh medium for 3 days or keep them constantly in doxorubicine for 3 days.
Thanks Agnieszka,
I got meanwhile also the suggestion of another Topo II Inh: Etopside (12.5µM) for 24h and 4 days in regular Medium to recover, just in case someone else reads the thread....
Ho long do you stain them ? I heard many different incubations at 37°C from 1h to oN....but all on different cell lines than MCF 7
Etoposide is also good idea. I stain MCF-7 cells over night in incubator, they stain nicely :)
increase number of passages of cultre. cells will undergo senescence and then stain for beta-Gal.
Nupur Agarwal it works only for normal cells. Cancer cells do not undergo replicative senescence, cos they have active telomerase.
Agnieszka, I was wondering how dense you cells are when you treat with Doxo ? I have a confluency of roughly 50% but looking at the whole plate I have cells grown in big islands, so very tight at some points in the center, while aoround the islands there's still space to grow.
I also seeded freshly cells and treated when I had islands of only 3/4/5 cells but I am afraid that as they do not have yet the exponential growth phase, doxo will not impact a lot.....
I am seeding MCF-7 cells in density of 10 000 cells/cm2. Then I give them 24h to attach to the surface of the plate. So more or less they have enough time to divide once. I am always adding the drug 24h after seeding. And I am always seeding them in the given density. Density and how much they are attached to the plate are crucial factors for susceptibility to the drug. Morover MCF by default are growing in clusters, between which is free space, so you can't do anything about that.
i totally agree that cancer cells do not undergo replicative senescence but they are subjected to stress induced senescence. B gal assay is applicable for types of senescence.
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