Hi everybody, I have a question in regard to apoptosis vs. necrosis.
I have cells from murine origin and I treated them with 2uM of Doxorubicin. This dose induces a massive apoptotic response and in fact, when I stain these cells with Annexin V I see an increased expression of this marker.
I also have the same cells exposed to 60C for 30 minutes (I am aiming to induce necrosis/cell death, perhaps different from apoptosis). When I stain the cells exposed to 60C they are positive for Annexin V and 7-AAD (a marker of cell death). However, the cells treated with Doxorubicin are Annexin V positive only (and 7-AAD negative). Therefore, I started to think that Doxorubicin is inducing an early apoptotic response and incubation of cells to 60C is inducing late/apoptosis necrosis. Based on this assumption, I collected the media from the cells exposed to the two different experimental conditions, I extracted the cfDNA and I ran a Bioanalyzer chip to see if I could observe any difference in the electropherogram.
Results: Cells Treated with Doxorubicin showed a majority of large DNA fragments. However, the cells exposed to high temperature showed a pattern rich of small fragments. I attach two examples here.
I am very confused because I was expecting small DNA fragments when apoptosis is involved and perhaps, longer DNA fragments when necrosis/cell death/late apoptosis is involved. This based on what is published in the literature.
Do you have any suggestion to explain this difference? Any insight would be greatly appreciated.
P.S. The Control (untreated cells) shows an electrophoretic pattern similar to the cells treated with Doxorubicin.
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