Jon I have used this reagent extensively with little trouble. You didn't say which compnay you're geting it from - I have used predominantly GIBCO BRL'sRED670 but also some of Pharmingen's. It is known the Cy5 can bind to Fc receptors (Carlton Stewart presented this at Lake Placid - don't know if he published it - Carlton can you tell us?). You can block with anti-Fc receptor (clone 2.4G2). Also have you done a proper titration. It might be you are using too much. If you took the manufacturer's suggested use you are for sure. GIBCO suggests to use 1ug/106 cells (although they do suggest titering it). We use 125ng/test (that's a 1:500 dilution and 25ul/test sample) on 106 cells! Titrate the reagent not for brightest staining but best signal to noise ratio. Larry Arnold Univ. of North Carolina At 05:52 PM 7/31/97 +0100, you wrote: > >Hello all, > >We are getting a lot of background fluorescence after using streptavidin Cy5 to >label biotinylated primary Mabs to mouse thymocyte surface markers. Can anyone >out there suggest a reason for this and how it can be minimised? Are there any >blocking steps that are needed for other biotinylated moieties on the cell >surface? Any thoughts would be much appreciated. > >Thanks in advance, >Jon >
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