why normal human fibroblast detached suddenly
Posted 02 April 2002 - 01:30 AM
Posted 03 April 2002 - 07:04 AM
Posted 03 April 2002 - 02:31 PM
If you were to leave the flask the detached cell layer will have cell outgrowths. (This is the best way to confirm that the cells are not dead.) It has even happened to me that the entire cell layer has rolled up into a ball !
However, once again if you let the ball settle you end up with a cellular out-growth.
I cannot say what it does to the phenotype of your cells, but my best advice is to subculture at between 80 and 90% confluence to prevent this from happening.
Posted 04 April 2002 - 03:34 AM
P.S. i still don't know whether the cells still live or not.because the culture medium became yellow,and I can't see any difference of the cells under microscope.maybe just the materials sent out by the cell after it died that tured the medium yellow.
Posted 04 April 2002 - 12:35 PM
1) an infection - possibly bacterial (generally the medium will become cloudy if this is the case) or mycroplasma (you can't see these and often they don't kill cells just make them behave abnormally). To test for mycroplasma you can get a staining kit from Sigma or a PCR kit from ATCC.
2) The cells were not feed for a prolonged period
3) ??? its just one of those cell culture things that happens sometimes.
Sorry I don't have any better ideas.
Posted 05 April 2002 - 06:42 AM
thank you for your help.I am a new comer in cell culture,so there are so many difficulties i meet.I still have one question,do you have any experience in synchronizing fibroblast cell in each cell cycle?
Posted 05 April 2002 - 07:38 AM
I also remember that you can collect some cell types in the M phase because they round up and are poorly attached to the substratum. You shake the medium to dislodge them and then recover them from the medium by centrifugation. I don't know how good this is though.
If you have any general questions on cell culture you are welcome to e-mail me at firstname.lastname@example.org. I have 12+ years experience with cell culture, mostly primary bone, cartilage and ligament fibroblasts but also with various connective tissue cell lines.