nowadays I am working on chicken kidney cell culture and CEF and i want to know how to know that these cells reach the hayflick limit and after this what i can do for these cells to make such kind of refreshment for it ?
You can detect cellular senescence using a B-galactosidase assay. Here is a paper talking about B-galactosidase and cellular senescence. http://genesdev.cshlp.org/content/24/22/2463.full
And a protocol paper:
http://www.springerprotocols.com/Full/doi/10.1007/978-1-59745-361-5_3?encCode=TU9NOjNfNS0xNjMtNTQ3OTUtMS04Nzk=
Or you just calculate it roughly. The Hayflick limit is 50 +/- 10 doublings.
When you have cells with a known doubling time, for example 48 hours, this means that your cells will start dying after around 100 +/- 20 days. You can see this easily using your microscope. The cells start to get apoptotic, e.g. they form apoptotic bodies and start to shrink. In addition, you can also use simple trypan blue staining to determine the number of living cells. An increasing number of dead cells is a clear sign for a dying culture.
That is a good estimate to follow, though I've had primary lines go up to 80 doublings. You can determine by trypan blue, but that is only for apoptotic cells it does not factor in the senescing cells that could stay in G0 for quite some time. Looking at both the beta galactosidase and trypan blue would give you a thorough observation.
80 doublings? Awesome....which cell line was it? And you are right: trypan blue is not the right choice for G0 cells.
Ah, well cells go through the cell cycle which have the growth phases known as G1 and G2. In G1, if the cell has DNA damage it does not continue to S phase and instead enters G0 which is a resting phase. This resting phase has a few outcomes, senescence is where the cell does not replicate but stays resting until the DNA damage or degradation is repaired or it can undergo apoptosis. G0 can also be a characteristic of terminal differentiation where the cell enters a quiescent state.
Here is some information about cell cycle control and such (I added the figure of the cell cycle from this paper as an attachment): http://www.bioscience.org/1999/v4/d/berndt/berndt.pdf
Oh and that cell line was human retinal pigmented epithelial cell (ARPE-19).
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