Apoptosis

[mat]

Hello,
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

[email protected]

[DevGrp]

With Eahy and GH3 cells we used between 100-1000uM H2O2 and were able to detect apoptosis using ELISA for DNA fragmentaion (see http://www.ncbi.nlm.nih.gov/entrez/quer ... s=10320817)
after 6hours. I wonder if you should look later - 24hr??
Can you see any apoptosis blebbing under the microscope?
To mean your dose seems high can you see any cell death? are all the cells undergoing necrosis?



That said I don't now anything about A549 cells so they could be less sensitive

[mat]

Hi,
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 don’t 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 don’t know where the problem is, ; but I am sure that I can’t continue for few months more.
If you can help me, please contact me.

Thanks a lot.

Mathieu kerbiriou

[DevGrp]

I must admit when assesssing apoptosis I tended to harest the cell from the supernatant (centrifugation)as some apoptotic cells become detached

Another possibility is to try a diferent inducer of apoptosis as a control

how about Camptothecin?

http://www.bdbiosciences.com/pharmingen ... osis.shtml