1) Why is spermine included in the Vindelov/detergent-based systems?
Spermine stabilizes the nuclei produced in this procedure and helps prevent
release of chromatin which will cause clumping.
2) Why do some people use detergents and others use ethanol
permeabilization?
Detergents are useful for releasing cells and nuclei from solid tissues, such as
tumor specimens. Detergents can also permeabilizeEthanol fixes the whole cell
in addition to permeabilizing it. This can be useful for some techniques where
other cytoplasmic antigens are detected with antibodies and you need whole
cells. In some cases the final CV of the DNA peaks varies with the
permeabilization method.
3) How does one make a buffer for eluting low MW DNA out of apoptotic cells?
If I remember correctly, Dr. Darzynkiewicz discussed this a few months ago and
you can search the archives for a more complete discussion. From his chapter in
"Techniques in Apoptosis, A user's guide" edited by Cotter and Martin, Portland
Press, London, 1996, he recommends:
1. fixing in ethanol (70%)
2. resuspending in 0.5 ml PBS
3. adding 0.2-1 ml of extraction buffer (0.2 M Na2HPO4, 0.1 M citric acid
buffer (pH 7.8)) - amount is inversely proportional to the degree of apoptosis.
4. incubate 5 min., centrifuge and stain for DNA followed by FACS analysis.
Best wishes,
Tony Bakke, PhD
Director, Flow Cytometry Lab
Oregon Health Sciences University
Portland, OR