Dr. Vladimir Krystof inquires about the method to extract DNA from apoptotic cells for gel electrophoresis to detect the "laddering". Some time ago we described a simple methodology for DNA extraction from apoptotic cells that can be combined with flow cytometry. The cells are fixed in 70 % ethanol, the cell pellet (after careful removal of ethanol) is then extracted with small volume (e.g. 50 ul) of high molarity Na- phosphate-citric acid buffer (0.2 M, pH 7.8). Under these conditions only low MW (fragmented) DNA is extracted from apoptotic cells. The cells are then centrifuged, the small volume of the supernatatnt buffer with the extracted DNA is sequentially treated with RNase and proteinase K and loaded on gel. Because only fragmented DNA is selectively extracted from the cells, the method is very sensitive. Furthermore, the cells from the pellet can be analyzed for DNA content by flow cytometry- apoptotic cells having fractional DNA content can be distingushed from nonapoptotic cells; the later display the cell cycle/ploidy distribution (Gong et al., Analyt. Biochem., 218: 314-319, 1994, also Current Protocols in Cytometry, 7.5.10) Zbigniew Darzynkiewicz Brander Cancer Research Institute New York Medical College 19 Bradhurst Ave. Hawthorne, NY 10532 tel: 914-347-2801 fax: 914-347-2804 http://www.geocities.com/z_darzynkiewicz
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