I need to get in on this, especially if there are answers that are not
posted. I use avidin CY5 all the time for confocal work and have a real
problem with fluorescence that appears when I use only CY5 that has
spectral characteristics like CY3???? I would kill for help on this.
We have no problem with nonspecific fluorescence using the appropriate
optics for CY5, just this goofy crossover light...
Thanks for any help.
Deb Berglund
Montana State University
On Fri, 1 Aug 1997, Larry Arnold wrote:
>
> 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
> >
>
>