Hi Karim, I use TO-PRO-3 extensively here as a dead cell discriminator. We have a couple of dual laser Caliburs and being able to get away from always using PI or 7AAD has freed up the other fluorescence channels for other markers. As for sensitivity, I did a lot of checking that TP3 labelled the same cells as PI and it is assumed that it works in the same way ie it will only enter cells with compromised membranes and will intercalate into the DNA helix. One thing that I did find though was that on our sorters where we use higher laser powers at 488nm, we are seeing some excitation of the TP3 there and emission from the primary beam. On the Calibur, the signal is slightly less intense than PI but this isnt really an issue. I would normally use a stock of about 1uM and add 10ul per ml. It is not normally necessary to RNase treat cells before using any DNA-binding dye as a dead cell discriminator as the dye should be excluded from the live cells. Hope that helps, Derek On Thu, 12 Oct 2000, Karim Vermaelen wrote: > 1. We'd like to do some 4-5 color analysis with dead cell exclusion in > FL4. Anybody has any experience with TOPRO-3 and sorts (Mol. Probes)? > How does it compare (sensitivity/specificity) with PI or 7-AAD? > 2. When doing dead cell exclusion, is it necessary to RNAse-treat cells > before applying the DNA-probe (like you would do for cell cycle > analysis); we're working with highly activated cells with high RNA > synthesis and we're concerned about possible uptake of the > nucleotide-probe by mRNA. > > Thanks a lot! > > Karim Vermaelen ************************************************************************ 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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