A simple calculation will show that steric hindrance cannot be a problem. Note that PE is about 4x the molecular weight of APC, and so is about 1.5x as large--switching to PE cannot possibly help if steric hindrance were the problem. (Which it is not). As Nicole alluded, most likely your problem is one of compensation (considering that the single-stained samples look OK). However, you should check to make sure that you have red-sensitive PMTs for the CY7APC. "Normal" PMTs simply do not have the sensitivity out there to work--getting the proper PMTs can increase signal to noise by an order of magnitude. We have made dozens of reagents conjugated to Cy7APC, with excellent results. In terms of "relative brightness", it is at least as good as fluorescein conjugated-antibodies. If you aren't seeing that kind of brightness, then you need to examine your optics. mr At 6:26 PM +0200 8/7/01, Karim Vermaelen wrote: >Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; > >Content-Transfer-Encoding: 7bit > >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 > >Content-Type: text/x-vcard; charset=us-ascii; > name="Karim.Vermaelen.vcf" >Content-Transfer-Encoding: 7bit >Content-Description: Card for Karim Vermaelen >Content-Disposition: attachment; > filename="Karim.Vermaelen.vcf" > >Attachment converted: MR Traveler:Karim.Vermaelen.vcf (TEXT/ttxt) (00035CC2)
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