Griffonia simplicifolia I isolectin as a functionally monovalent probe for use in flow cytometry.
The inherent tendency of lectins to agglutinate cells has limited their use as reagents for the detection of carbohydrate groups on cell surfaces by flow cytometry. In the current study, we demonstrate a method for the use of a fluoresceinated tetrameric isolectin (Griffonia simplicifolia I-A3B, FITC-GS I-A3B) as a functionally monovalent, nonagglutinating probe in flow cytometry. This isolectin contains three A subunits and one B subunit. Both types of subunits bind alpha-D-galactopyranosyl (alpha-D-galp-) end groups with similar affinities; however, the A subunits have a 1,000-fold greater affinity for N-Acetyl-D-galactosamine (GalNAc) than does the B subunit. The addition of low (1-2 mM) concentrations of GalNAc to the FITC-GS I-A3B isolectin results in blockage of the three A subunits without significantly affecting the B subunit; this yields a functionally monovalent probe for the detection of cell surface alpha-D-Galp end groups. This approach has been used to examine two types of cells: Ehrlich ascites tumor cells and rat alveolar macrophages, both of which are known to express cell surface alpha-D-Galp end groups. Lectin binding, as determined by number of positive cells and fluorescence intensity, was dependent upon concentration of the lectin and haptenic sugar. Specificity of the staining was demonstrated by the ability of methyl alpha-D-galactopyranoside (Met alpha-D-Galp) to abolish the binding of the lectin to the cells. Elimination of both GalNAc and Met alpha-D-Galp from the staining solution resulted in agglutination of the cells, indicating that the A subunits were active in the absence of GalNAc.
J P
JP
McCoy
N
N
Shibuya
M C
MC
Riedy
I J
IJ
Goldstein
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis / Q000473:pathology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006241:Haptens / Q000032:analysis; D037102:Lectins / Q000032:analysis; D008264:Macrophages / Q000032:analysis / Q000166:cytology; D051379:Mice; D037121:Plant Lectins; D011650:Pulmonary Alveoli / Q000032:analysis / Q000166:cytology; D051381:Rats; D013194:Staining and Labeling
D002241:Carbohydrates; D005456:Fluorescent Dyes; C045475:Griffonia simplicifolia lectins; D006241:Haptens; D037102:Lectins; D037121:Plant Lectins