Regarding my message below. The problem was due to overcompensation and has been corrected. Thank you for all your assistance and I apologize for being premature. Jose "Jose A. Stoute" wrote: > Dear all, I am having a problem with the use of three color reagents for > immunophenotyping and measurement of leukocyte surface markers. > I currently use CD14 FITC and CD45PECy5 from Pharmingen to identify my > different cell populations. In addition, I use PE-conjugated antibodies > for the detection of surface markers of interest. I noticed that > CD14/CD45 alone works beautifully but whenever it is used in combination > of the PE-conjugated antibody against the surface marker the CD45 signal > is decreased or completely eliminated. This does not happen with the > corresponding isotype control which is also PE conjugated. I suspect > there may be some energy transfer going on between PE and PECy5 which > perhaps I should have anticipated but I am not sure. > I have the following questions: > 1. What is going on? > 2. How can this be remedied or prevented without having to switch to > another fluorochrome? > > I thank you for any assistance you can give me. > > Regards, > > -- > Jose A. Stoute, MD > Unit 64109, Box 401 > USAMRU-Kenya > APO AE 09831-4109 > e-mail: stoutej@net2000ke.com > Nairobi Tel 254-2-729303, Fax 254-2-714592 > Kisumu Tel 254-35-22942, Fax 254-35-22903 > Electronic Fax Service: 1-630-214-2008, 1-917-463-0373, 1-917-477-6048 -- Jose A. Stoute, MD Unit 64109, Box 401 USAMRU-Kenya APO AE 09831-4109 e-mail: stoutej@net2000ke.com Nairobi Tel 254-2-729303, Fax 254-2-714592 Kisumu Tel 254-35-22942, Fax 254-35-22903 Electronic Fax Service: 1-630-214-2008, 1-917-463-0373, 1-917-477-6048
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