Hello Flowers, I have a user who wants to sort CD3 and CD33 positive cells from stained leukocytes for DNA extraction. They are looking to recover at least 500,000 cells each. The DNA will be used for PCR analysis of polymorphisms. One concern is that the sorter for some reason may not be available on the day we receive the samples necessitating the fixation of the stained samples. As some of you may know, DNA is very difficult to extract from formaldehyde fixed cells. We have tried 50% and 70% ethanol fixation with mixed results. The biggest problem is the loss of cells after ethanol fixation. Even after careful recovery of the samples from the ethanol the cells are fragile to the point where they slowly lyse even at 4oC. There is further lysing during the sorting which causes the flow cell and the nozzle to eventually clog (I am sorting on a FacsAria.) Although I prefer to sort live cells for DNA extraction, is there a protocol out there that will fix cells in a manner so the DNA can readily be extracted for PCR or other molecular biology assays? I have tried others in the past such as methanol, gluteraldehyde, and Permeafix with less than promising results. Any help with this will be sincerely appreciated! Earl Earl A. Timm, Jr. Research Associate Laboratory of Flow Cytometry Roswell Park Cancer Institute Buffalo, New York 14263 earl.timm@roswellpark.orgReceived on Wed Oct 13 15:38:00 2004
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