Hello,
Anybody know if adherent cells (I am using U2OS, but any general information is helpful) progress through the cell cycle when detached from the dish (I use trypsin).
I want to start sorting some U2OS and then put them back in culture.
I would like to see if/how they recover from the stress caused by the sorting and how long does it take to the cell to recover and progress.
Thanks for your help!
Suspended U2OS cells remain viable and they will re-adhere and keep growing but their growth is likely to be affected while they remain in suspension.
This paper shows some methods to confirm that U2OS cells are going through their cell cycle:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842991/
The cell cycle length of U2OS cells is ~18–20 h, and so a 48-h time course would be expected to have only ~2.5 cell cycles.
Thanks, I would study the cell cycle progression. So, after sorting cells in G1, I need to re-seed the sorted cells and then wait hours before trypsinise them again and study early S- Mid-S or late S..
Laurence Stuart Dawkins-Hall
in theory I would like to study the S phase right after seeding the G1 cells, so I think 48h is too much and the risk I see are two:1. loose the synchronisation after G1 sorting ( a subset of population could proliferate quicker)
2. to study NOT the first cell cycle right after sorting (where I think the cells should more synchronously move through the cell cycle) but rather the second one.
Serum has a large role in regulating the cell cycle. If you remove serum from ur media after trypsin treatment for suspension ur cells would freeze at the position in the cell cycle when it last received growth factors from serum. Attachment may not be a crucial factor for cell lines. And it should not affect ur sorting g1 cells. It's difficult not to use antibiotics after sorting but also make sure u use fresh serum for your revival of cells. Attachment depends on serum factors. And the time these cells were out side the incubator without the bicarbonate buffering system. For a while I had used glass plates with out poly lysine coating. So they don't feel shy attaching to any surfaces..
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