I have used lectins in flow. Titration is the name of the game. You will reach a level of surface staining without crosslinking all of the cells if you go out far enough with your dilutions. I used them to quantitate levels of sugars expressed at the cell surface. For this application, flow provided excellent quantitative results. Direct labels work much better. Biotin-Avidin usually requires a wash step and can promote clumping- don't do it. You will have these reagents titrated so far out that it really isn't necessary. I realize that biotin is often the only label available for many lectins. Also keep everything on ice during staining and use at least 1% protein in your staining buffer. Here is a reference from a previous life: Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7190-5. PMID: 8692967; UI: 96293498 Good luck, let me know if you have any further questions. Steve McClellan Beckman Coulter ----- Original Message ----- From: Carolyn Jefferiss <prscmj@bath.ac.uk> To: cyto-inbox Sent: Thursday, June 22, 2000 5:19 AM Subject: lectins > > Dear Flow People, > Please, can anybody give me some help with methods for using conjugated > lectins such as soybean agglutinin for FACsing either as a > directly-labelled conjugated (SBA-FITC) or indirectly (SBA-biotin then > streptavidin-FITC)? How, for instance, does one label cells individually, > let alone analyse or sort such cells without clogging up the works? > Thank you for any help forthcoming. > Yours sincerely > > Carolyn Jefferiss > > Carolyn Jefferiss Ph. D. > Pharmacy and Pharmacology > University of Bath > Claverton Down > Bath BA2 7HY > >
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