title: PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205.
abstract: DEC205/CD205, an endocytic receptor of C-type multilectin, is expressed highly in dendritic cells (DCs). DEC205 was shown to efficiently deliver vaccine antigens in surrogate ligands to the antigen processing and presentation machinery of DCs, which resulted in the development of DC-targeted vaccines employing anti-DC monoclonal antibodies (mAbs). During our studies to characterize a variety of anti-DC mAbs including anti-DEC205 by flow cytometric analysis, we discovered that a secondary anti-immunoglobulin antibody conjugated with PE-Cy5.5 bound strongly to the cells expressing mouse DEC205 (mDEC205) without incubation of a primary anti-mDEC205 mAb. In the present study we demonstrate that various antibodies and streptavidin conjugated with PE-Cy5.5 bind to the mDEC205-expressing cells including CHO, KIT6, and HEK293 cells. The interaction between the PE-Cy5.5 conjugates and the cells expressing mDEC205 appears distinctive, since none of the PE-Cy5.5 conjugates bind to the cells that express human DEC205 on surface. Besides, only PE-Cy5.5 conjugates bind strongly to mDEC205-expressing cells; PerCP-Cy5.5, APC-Cy5.5, and Cy5.5 conjugates bind weakly; PE, PE-Cy5, Cy5, FITC, or Alexa488 conjugates do not bind to mDEC205-expressing cells. Therefore the use of PE-Cy5.5 conjugates, widely utilized in multicolor flow cytometry, requires precaution against nonspecific binding to mDEC205-positive cells.
authors: Laboratory of Cellular Physiology and Immunology and Chris Browne Center for Immunology and Immune Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. parkc@rockefeller.edu
authors: Chae Gyu
authors: CG
authors: Park
authors: Anthony
authors: A
authors: Rodriguez
authors: Ralph M
authors: RM
authors: Steinman
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001667:Binding, Competitive / Q000276:immunology; D016466:CHO Cells; D002232:Carbocyanines / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D003713:Dendritic Cells / Q000276:immunology; D005434:Flow Cytometry; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D015778:Minor Histocompatibility Antigens; D011485:Protein Binding / Q000276:immunology; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013045:Species Specificity; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; C093569:DEC-205 receptor; D018796:Immunoconjugates; D037181:Lectins, C-Type; D015778:Minor Histocompatibility Antigens; D011956:Receptors, Cell Surface; D019809:Streptavidin