Apoptosis in adherent and floating cells - (Oct/09/2008 )

Hi!
I'm trying to show DNA laddering in cultured tumor Leydig cells after induction of apoptosis. I already set up concentrations and time by observing cells stained with HOESCHT 33342 (the vital probe). My question araised from these observations: the apoptosis process is not synchronized (obviously), so that not all the adherent cells show apoptotic nuclei. On the other hand, I argue that floating (=dead cells =apoptotic bodies) have fragmented DNA.
In the very first experiment I pooled adherent and floating cells, extracted DNA without finding any ladder
Should I keep adherent DNA separate from that from floating cells? Would I have more probabilities to see DNA laddering??

please help me! nobody can help me here!!

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I dont know this will help you or not, but I think it is better not to include dead cell for your study, becuase floating cells are dead alreday, not neccesary apoptotic body. its just a junk. So, if I were you I will only use DNA from aderent cells

also,
when you did your first experiment using DNA from adherent and non-adherent cells, what kinds of result you got?
DNA was full lenght? or alreday only fragment?
if DNA is still full length, your treatment might not be inducing apoptosis enoght, you might need to increase concentration, or increase incubation time.
if DNA is alreday only small fragment, your treatment might kill cells completely before you starts assay, so you might need to adjust treatment (reduce concentration) or reduce incudation time.

if majority of cells are apoptoticm, DNA ladder is quite visible. but also be carefull not to damage/degrade DNA sample after extraction.

Good luck

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