Hi Paulette, You are right in that there can be significant cell loss during BrdU staining - I always recommend that to get 20,000 useful events, you need to start with 10x that number (not that 90% will be lost but if there is a pellet you can monitor cell loss more easily). If the cells have been ethanol fixed, you need to be quite careful spinning them; I always spin them quite hard - you probably wont induce any cell damage. The acid-denaturation method does involve a number of centrifugation steps so be careful with removing the supernatant. There are some downloadable protocols on my website: http://sci.cancerresearchuk.org/axp/facs/davies/CycleBrdU.html Good luck! Derek > We have recently started trying to use BrdU/PI flow to study the cell >cycle characteristics of embryonic mouse brains. We have had some success >in obtaining single cell suspensions but are having real problems with >cell loss during BrdU staining which we think is related to HCl treatment >to denature the DNA (could this be lysing these fragile cells?). 70% >ethanol fixed cells treated with 2N HCl for 30mins show good BrdU staining >under the microscope but are really very few (90%+ lost) Has anyone done >such an analysis? Does anyone have a suitable protocol? Or know anyone who >does? Or any good ideas? We look forward to any helpful suggestions. >Paulette Zaki and Ben Martynoga Department of Biomedical Sciences >University of Edinburgh >Get faster connectionsİ-- switch toİMSN Internet Access! ><http://g.msn.com/8HMOEN/2017>Click Here ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 London Research Institute, e_mail: derek.davies@cancer.org.uk Cancer Research UK mobile: 07790 604112 44 Linolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux *************************************************************************
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