IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

DNA-based assay of apoptosis needed

Robert W. St. G. Fisher, IV fisher at radonc.unc.edu
Thu Feb 6 11:17:38 EST 1997


there are a couple of ways to assay for the DNA fragmentation that
occurs during apoptosis.

1) (probably the easiest)  extract DNA from your apoptotic cells (if
doing it in TC don't forget to spin down the media to get the floaties),
and run it out on a 2% agarose gel and look for multiples of the 180 bp
fragments (DNA ladders).
2) Boehringer Mannheim sells a kit that detects histone-associated DNA
fragments. I have heard that the first version of the kit may detect
necrosis too, though.  I have tried the kit, and it works fairy well for
my samples.
3) do flow cytometry.  do a whole cell prep and stain with propidium
iodide.  look for a sub G1 peak.  those will be the apoptotics. 
however, you will probably need to optimize the assay timewise so that
you get the cells before they break apart (when this happens you get a
big sub G1 smear).
4) use the TUNEL assay and analyze by flow.  there are a lot of
manufactuers that make kits for this too.  


methods 1 and 3 are the quickest and easiest (IMHO).  2 and 4 may give
you better numbers...it kinda depends on what kind of samples you're
analyzing...if you're trying to do it in tissue slices, the TUNEL assay
is your best bet.   For TC samples, the first three should be
sufficient.  

You will probably wind up doing more than one assay anyway, to satisfy
yourself (and everyone else) that what you're seeing is actually
apoptosis.......

Just my 2 cents worth......

Robert W. Fisher
fisher at radonc.unc.edu



More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net