I was wondering whether someone has a protocol to get decent cell cycle synchronization of normal human keratinocytes in vitro. I had done a double thymidine block with HaCaT cells but I am not sure whether this is appropriate for non-immortalized primary keratinocytes. And what are the dynamics of the synchronization: when do cells enter what phase of the cell cycle?
Hello
Kindly look at links here
Good luck
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003419
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056451/
Take a look at Joany Jackman and Patrick O'Connor's protocol paper: Methods for Synchronizing Cells at Specific Stages of the Cell Cycle. DOI: 10.1002/0471143030.cb0803s00
It does a rather good job of both explaining the cell cycle, explaining different ways to stop it at different points, and providing specific, fairly reliable protocols for a number of different mechanisms to achieve cell cycle arrest.
Thank you. Most of the publications use immortalized or cancer cell lines. Primary epithelial cells seem not to be mentioned, used or considered. I assume some of the methods in the Jackman paper should work on primary cells as well without affecting their health, causing senescence or any other undesired reaction?
senescence could occur , but you won't know until you try it Thomas.
-julie
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