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
>