Apoptotic DNA fragmentation analysis

Francis Chun-wai CHAN cwaichan at cuhk.edu.hk
Sat Jun 21 13:29:44 EST 1997

In article <plxjha.2.0010999B at pln1.life.nott.ac.uk>,
plxjha at pln1.life.nott.ac.uk (John Harris) wrote:

> We are interested in extracting DNA from apoptotic cells (primary 
> cultures of cerebellar granule cells). We have been using a 
> tris/EDTA/Triton-X-100 extraction followed by phenol/chloroform extraction 
> proteinase-K treatment, ethanol precipitation and RNase treatment, all fairly 
> standard stuff!! When the DNA samples are run on the gel however we get
> patterns in both our control and apoptotic cell lanes. Control cells 
> have a normal morphology and Hoechst 33258 staining of their nuclei shows an 
> even distribution of chromatin, distinct from the abundant apoptotic bodies 
> seen in our apoptosis positive cells. Does anyone have experience with DNA 
> extraction from granule cells? Any suggestions as to why we get a DNA ladder 
> pattern in our 'control' cells would be welcome and any suggestions as to how 
> we go about getting rid of it would be even more welcome.

If the control cells are cultured in prolonged time  (e.g. more than  2
days) without enough serum, the certain population of cells (usually 10%
per days) may be spontaneously undergone apoptosis.

Francis Chun-wai CHAN
Dept of Ortho. & Trauma, 
cwaichan at cuhk.edu.hk

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