Dear Dave, Of course you are right. If we exlude the dead cells from the analysis the FL3 staining won't influence the PE stain (because it doesn't exists on live cells) consequently we don't need FL2-FL3 compensation, namely PI or 7AAD will work equally. I just wanted to point out in my message to Rachel what you say on your web page too: "A very useful alternative to propidium iodide for live/dead discrimination is 7-amino actinomycin D (7-AAD). The most significant advantage is the longer wavelength emission of 7-AAD that makes it easier to use PE as a surface marker at the same time." As you can see you have fallen in the same trap in the above sentences or indeed the context is not good. Of course we have to separate DNA staining for live/dead cell discrimination (where does not matter either PI or 7AAD is used) from the surface+DNA labeling for phenotype+ploidy measurements where your second sentence is absolutely true and we must not to mix up the two things. At this point I have to apologize for the misleading message to Rachel and thank you for alert. Best regards, Pal On hétfő, december 17, 2001, at 03:33 , DAVID M CODER wrote: > Either PI or 7-AAD will work. Moreover, with either DNA-binding dye, > compensation in only direction is needed: You must make sure that > PE-positive cells do not appear to be 7-AAD or PI-positive. > Compensation 7-AAD or PI into PE positive space is unnecessary as those > cells are eliminated from further analysis. The attached images > illustrate compensated vs. uncompensated PE. > > For details see web page that I developed when I was directing the lab > at the University of Washington: > http://nucleus.immunol.washington.edu/Research_facilities/Apps/7aad.html > > Dave > ---------------- > David M. Coder, Ph.D. > Consulting in all aspects of cytometry. > > email: d_coder@msn.com > tel./messages: 206-499-3446 > > ----- Original Message ----- > From: Jáksó Pál > To: Cytometry Mailing List > Sent: Monday, November 26, 2001 11:21 PM > Subject: RE: 7AAD PROTOCOLS > > Dear Rachel, > > 7AAD is may be better in some application because of the lower > crosstalking to the FL2 channel. Accordingly you can use it together > with PE stains. PI is very hard to compensate from the FL2. But if you > use only FITC as second stain (and nothing else) you can use both 7AAD > and PI for dead cell labeling. > > Pal Jakso > > University of Pecs > Faculty of Medicine > Dept. of Pathology > 7643, Hungary > 12. Szigeti str. > > > While this subject has come up: it seems like both 7AAD and propidium > iodide > can be (and are) used for detecting dead or apoptotic cells - are they > interchangeable ? If people have distinct preferences, what is the > difference ? > > thanks, > Rachel > >
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:43 EST