I'm also helping a colleague use Draq5. We got respectable or should I say recognizable histograms on an analytical scale. However, the only use for this dye is presumably preparative work. When we scaled up for a preparative run (more cells at a higher concentration) the histograms were less impressive--basically a big fat ugly peak with a shoulder to the right where S and G2 should have been. Does anyone have experience with this dye on a prepartive scale, that is cell #, cell concentration and dye concentration. Were the histograms as good as on the analytical scale? Did you get enough cells to re run in order to check for purity? Sincerely, Dan Rosson Dan Rosson Ph.D. Lankenau Institute of Medical Research 100 Lancaster Ave. Wynnewood, PA 19096 www.limr.org. -----Original Message----- From: Derek Davies [mailto:daviesd2@cancer.org.uk] Sent: Thursday, March 07, 2002 10:04 AM To: cyto-inbox Subject: Re: DRAQ5: may be an alternative for Hoechst 33342 if I have only 488nm argon laser? Hello Pal, Yes, we have used DRAQ5 here as a vital DNA dye. You are right that on excitation by a 488 Argon laser it emits in the far red so on a FACScan or single laser FACS Calibur it will be detected in FL3. I have attached a small graphic file to show Jurkat cells unstained and stained with 5uM DRAQ5 for 2mins. I have run these on a Calibur with log amplification for both FL2 and FL3 to show that no compensation is needed to remove DRAQ5 signal from the FL2 channel. Obviously if you were using it for quantitative DNA assessment, you would use linear amplification. There would still be some compensation needed to remove bright PE signals from FL3. We have used DRAQ in combination with GFP and Pyronin Y so it can be done! Derek On Wed, 6 Mar 2002, Jáksó Pál wrote: > I read a post from Derek Davis in which DRAQ5 a far red emitting DNA > stain from Biostatus was recommended for DNA staining for flow cytometry > without permeabilization of living cells. I would like to ask about your > experiences with this new DNA dye. > I intend to measure surface immunostainings in conjuctions with DNA > content using 488 nm argon laser. At the Biostaus website is stated that > using together with FITC compensation is not necessary and DRAQ5 can be > also used with PE. ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Cancer Research UK, e_mail:derek.davies@cancer.org.uk London Research Institute, mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux *************************************************************************
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