From: Art Roberts (robertar@UMDNJ.EDU)
Date: Thu Oct 22 1998 - 15:12:40 EST
Hi Fellow flow-ers, I am trying to detect an increase in Fas-ligand expression on lymphocytes of specific TCR-Vbeta types. To this end, I want to do 2-color analysis using indirect staining for each Vbeta (utilizing GAM-FITC and a mIgG block of any unoccupied anti-IgG), followed by biotinylated anti-FasL and avidin-PE. When I did this, however, I got no FasL +ity at all. So I decided to run C1R cells, which express a respectable level of FasL, as a 1-color positive control. Indirect staining with anti-FasL (Oncogene #AM29) and GAM-FITC gave a 2.5X shift (on a linear scale, with all due respect to Ray Hicks and Howard Shapiro) in mean fluorescence, but when I used the biotinylated anti-FasL (MBL #D041-6 hamster IgG) and avidin-PE, I could not detect any shift in the FL2 signal. Without a detectable signal using the biotin conjugate, my 2-color analysis is useless, but it seems that the intensity of fluorescence produced by the singly bound avidin-PE is just too dim to detect at the low level of FasL expressed. My question: Is there any way to enhance the detection of the small amount of biotinylated antibody that is bound? Thanks in advance for any advice. Art Roberts Dept. of Medicine Robert Wood Johnson Medical School email: robertar@umdnj.edu phone: 732-235-7790 Fax: 732-235-7792
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