high levles of cell death in PC12 cells - (Jun/09/2008 )
Hi,
I am working with the PC12 cell line and have stably transfected them with the gene i am working on. I treated my cells with hydrogen peroxide and then did FACS analysis to measure levels of apoptosis. I found that both my transfected and untransfected control cells had a 20% apoptosis rate without any treatment at all. This seems abnormally high; how would i explain this? i have repeated the expt 3 times and each time i get around 20% basal death; the cells are not serum starved.
-daph-
QUOTE (daph @ Jun 9 2008, 08:12 PM)
Hi,
I am working with the PC12 cell line and have stably transfected them with the gene i am working on. I treated my cells with hydrogen peroxide and then did FACS analysis to measure levels of apoptosis. I found that both my transfected and untransfected control cells had a 20% apoptosis rate without any treatment at all. This seems abnormally high; how would i explain this? i have repeated the expt 3 times and each time i get around 20% basal death; the cells are not serum starved.
I am working with the PC12 cell line and have stably transfected them with the gene i am working on. I treated my cells with hydrogen peroxide and then did FACS analysis to measure levels of apoptosis. I found that both my transfected and untransfected control cells had a 20% apoptosis rate without any treatment at all. This seems abnormally high; how would i explain this? i have repeated the expt 3 times and each time i get around 20% basal death; the cells are not serum starved.
If you treat the PC12 cells with H2O2 don't you expect some oxidative damage and cell death? If you had cells "not treated with H2O2" FACSed you may know this.
-cellcounter-
QUOTE (cellcounter @ Jun 10 2008, 05:41 AM)
QUOTE (daph @ Jun 9 2008, 08:12 PM)
Hi,
I am working with the PC12 cell line and have stably transfected them with the gene i am working on. I treated my cells with hydrogen peroxide and then did FACS analysis to measure levels of apoptosis. I found that both my transfected and untransfected control cells had a 20% apoptosis rate without any treatment at all. This seems abnormally high; how would i explain this? i have repeated the expt 3 times and each time i get around 20% basal death; the cells are not serum starved.
I am working with the PC12 cell line and have stably transfected them with the gene i am working on. I treated my cells with hydrogen peroxide and then did FACS analysis to measure levels of apoptosis. I found that both my transfected and untransfected control cells had a 20% apoptosis rate without any treatment at all. This seems abnormally high; how would i explain this? i have repeated the expt 3 times and each time i get around 20% basal death; the cells are not serum starved.
If you treat the PC12 cells with H2O2 don't you expect some oxidative damage and cell death? If you had cells "not treated with H2O2" FACSed you may know this.
Sorry, my post must have been confusing. I am seeing 20% death in cells that have not been treated with hydrogen peroxide which seems a bit high
-daph-
QUOTE (daph @ Jun 10 2008, 06:28 PM)
QUOTE (cellcounter @ Jun 10 2008, 05:41 AM)
If you treat the PC12 cells with H2O2 don't you expect some oxidative damage and cell death? If you had cells "not treated with H2O2" FACSed you may know this.
Sorry, my post must have been confusing. I am seeing 20% death in cells that have not been treated with hydrogen peroxide which seems a bit high
I have not worked with PC12 cells in particular, but following themes are common.
1. Over-confluent
2. Over-trypsinization
3. Submitting for flow much in advance of the procedure, sample not kept on ice
4. Not optimum dose of serum
5. Forceful -damaging pipetting
6. Bad or untested batch of serum
7. Bad media
8. Bad culture conditions (temp, humidity, CO2 level)
9. Non-ideal flasks, flasks with less aeration.
10. Long periods of culture without splitting
Oh, so many things can go wrong with cell culture that can result in cell death / apoptosis. If you are a veteran of cell culture, I suggest you check with FACS facility as to how they are treating your cells (time before processing/storage etc). If that is fine, I can't think of anything general.. PC12 Gurus can jump in.
-cellcounter-