Hi Martha, THere are a number of things to bear in mind with the BrdU technique. One is that it is only true to say that labelled cells are in S phase when the pulse with BrdU is short. The longer it is the more likely that labelled cells will have passed through S into G2 and even back round to G1 again. In general a pulse of 30-60 minutes will give a reasonable percentage of labelled cells. An 8 hour pulse will detect cells that were near the end of replication at the beginning of the pulse and these will have continued during the time BrdU was present to end up elsewhere. The BrdU technique is quite versatile as it allows you to address different questions depending on your labelling and/or sampling time. If, after 8 hours, you see low levels of incorporation and you are sure that your population is expanding, then there may be a technical problem. If you are using the acid denaturation method, you must be quite stringent with your washes after HCL; I find that a minimum of three PBS washes after spinning the acid off is required - you need to make sure that your primary antibody isnt denatured! There is also a balance between unwinding enough DNA to expose the BrdU to the antibody and not doing too much so that the resolution obtained with your DNA dye is lost. Too high a molarity or too lonmg in acid will lead to loss of DNA histogram resolution. I normally find that 15-30 mins in 2N HCL is a good starting point. Hope that helps a bit! Derek On Tue, 10 Apr 2001, Martha Mesa - Microbiologia wrote: > In my laboratory, I am studying the effect of some compounds on tumoral > cell cycle by PI staining. In order to know if the antitumoral effect > observed is due to a decrease in DNA synthesis I am trying to set the > BrdU incorporation assay, by I have several questions; some of them > are: > > 1. For cells with doubling time of 22 (k562) and 72 hours (KG1a), how > long must be the pulse? I have used BrdU 10 uM for 8 hours; I have > only obtained low intensity in fluorescence some times. > > 2.Some protocols recommend more HCl. I have used 2M, 3M and 4M. With 4M > the BrdU signal improves but phase resolution with PI disappear. I > would like to > know the effect in each phase; how can I obtained > that? > > Thanks in advance, > > Marta Mesa > > ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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