Karim, I guess the first question I have for you is what instrument are you using. Next, what are the filter sets/dichroics. Finally, have you tried different fluorochrome combinations to eliminate steric hindrance of the antibodies (perhaps changing one to a PE conjugate since this is also a fairly large protein). Randy T. Fischer NIH/NIAMS Autoimmunity Branch Building 10, Rm 6D57 9000 Rockville Pike Bethesda, MD 20892 (301) 594-3537 fischer1@mail.nih.gov > ---------- > From: Karim Vermaelen > Sent: Tuesday, August 7, 2001 12:26 PM > To: Cytometry Mailing List > Subject: Phar Red Blues > > <<File: Karim.Vermaelen.vcf>> > Hi, > Has anyone ever tried to co-stain double pos. cells with one marker in > APC, the other in APC-Cy7? We routinely costain murine MHCII and CD11c. > Now we need those markers in APC and APCCy7 resp. > We use a rat anti-mouse MHCII and a biotinylated hamster anti-mouse > CD11c, followed by APC-goat anti-rat (Caltag) and streptavidin-PharRed > (Pharmingen). > When used separately, both colours are reasonable (although I always > found PharRed to be quite disappointing). However when used together on > the cells coexpressing classII and CD11c, both signals from those > secundary reagents collapse to near oblivion... > Could there be steric hindrance? Quenching? Or something stoopid I'm > missing? I'd love to hear suggestions or anyone with similar > observations! > Karim > >
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