Posted 17 March 2005 - 02:42 AM
I have tried to induce a549 cells apoptosis with H2O2 for 4 months but I have no result! I detect a549 dna with ethedium bromide on agarose gel but I have no dna fragmentation!
My protocol is:
- cell culture on flask 75 cm3
- add H2O2 30% (5M, 1M, 100mM, 10mM final !!!)
- after 6 hours, wash cells whith PBS 1X and trypsination
- centrifugation at 2400 rpm 10 minutes and extraction of dna (CH3COONH4, ethanol 100%) from the pellet
- gel electrophoresis and revelation U.V.
My question is: can you send your protocol to me if you have succed this work ? At least, have you some idea or request to help me ?
Thank you a lot
Posted 17 March 2005 - 06:57 AM
Hope that it helps!
Posted 17 March 2005 - 07:35 AM
badcell, on Mar 17 2005, 07:57 AM, said:
Hope that it helps!
First, thank you for responding me.
I have already tested [H2O2]=900, 150, 400 10-6 M and 4mM during 16, 24, 48 and 72 hours. No result ! So I stopped kinetics and I increased the concentration (5M, 1M, 500mM, 100mM, 50mM and 10mM) for an incubation of 6 hours. No fragmentation ! So, I changed the solution of H2O2 and I repeated the manip. I checked the cells before the extraction and only few cells were in the supernatant. So, most of the cells donít undergo necrosis, but always no fragmentation.
I wonder if the cells undergoing apoptosis quickly loose their ability to stick the bottom of the flask or not ?
I donít know where the problem is, ; but I am sure that I canít continue for few months more.
If you can help me, please contact me.
Rq: I use a gel of agarose of 1,5%
Thanks a lot and good luck!
Posted 17 March 2005 - 05:29 PM
By my experience, cells in the early stages of apoptosis are usually adherent and don't float until they are in the late stages of apoptosis.
DNA laddering is supposed to be [mostly] dependent on activated caspase-3, so if A549 cells don't express active caspase-3 [or if it's not activated in response to H2O2 treatment] you might not see laddering. Also, I don't think DNA laddering is the best way to detect apoptosis.
Have you considered other apoptosis assays? There's loads to choose from, but for the detection of early-stage apototic cells, I find that Annexin-V staining is usually the most reliable indicator.