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
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