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Apoptosis


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#1 maldou

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Posted 17 March 2005 - 02:42 AM

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

mathieu.kerbiriou@univ-brest.fr

#2 badcell

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Posted 17 March 2005 - 06:57 AM

Are your cells dying? Have you been able to detect cell death in your experiment by other means (at least by seeing the cells swelling and getting off the plate after incubation with H2O2). If the answer to this is yes, then it may be just a problem of detection. Sometimes the ladder is not that strong and is difficult to visualize with ethidium bromide (for some time, I did label my DNA with 32P to see the ladder). Which concentration of agarose are you using in your gel? I couldn't see the ladder in agarose (using another cell line and pro-apoptotic stimuli) and I was able to see it in TreviGel, another type of matrix (http://www.trevigen.com/trevigel.php).
Hope that it helps!
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)

#3 maldou

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Posted 17 March 2005 - 07:35 AM

Are your cells dying? Have you been able to detect cell death in your experiment by other means (at least by seeing the cells swelling and getting off the plate after incubation with H2O2). If the answer to this is yes, then it may be just a problem of detection. Sometimes the ladder is not that strong and is difficult to visualize with ethidium bromide (for some time, I did label my DNA with 32P to see the ladder). Which concentration of agarose are you using in your gel? I couldn't see the ladder in agarose (using another cell line and pro-apoptotic stimuli) and I was able to see it in TreviGel, another type of matrix (http://www.trevigen.com/trevigel.php).
Hope that it helps!

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 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!

Mathieu kerbiriou

#4 Hooly

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Posted 17 March 2005 - 05:29 PM

I've used the Oncogene Suicide-Track DNA Ladder kit for this. What I see [in the same cell line] is that different treatments give different results, some give very strong DNA ladders some give very weak ladders, but all of which can be quite difficult to detect by agarose gel/EtBr. So it might depend on the particular apoptotic pathway you are stimulating with your H2O2 in this cell line, or on the detection method [as badcell says].

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.




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