title: Cell surface antigen profiling using a novel type of antibody array immobilised to plasma ion-implanted polycarbonate.
abstract: To identify and sort out subpopulations of cells from more complex and heterogeneous assemblies of cells is important for many biomedical applications, and the development of cost- and labour-efficient techniques to accomplish this is warranted. In this report, we have developed a novel array-based platform to discriminate cellular populations based on differences in cell surface antigen expressions. These cell capture microarrays were produced through covalent immobilisation of CD antibodies to plasma ion immersion implantation-treated polycarbonate (PIII-PC), which offers the advantage of a transparent matrix, allowing direct light microscopy visualisation of captured cells. The functionality of the PIII-PC array was validated using several cell types, resulting in unique surface antigen expression profiles. PIII-PC results were compatible with flow cytometry, nitrocellulose cell capture arrays and immunofluorescent staining, indicating that the technique is robust. We report on the use of this PIII-PC cluster of differentiation (CD) antibody array to gain new insights into neural differentiation of mouse embryonic stem (ES) cells and into the consequences of genetic targeting of the Notch signalling pathway, a key signalling mechanism for most cellular differentiation processes. Specifically, we identify CD98 as a novel marker for neural precursors and polarised expression of CD9 in the apical domain of ES cell-derived neural rosettes. We further identify expression of CD9 in hitherto uncharacterised non-neural cells and enrichment of CD49e- and CD117-positive cells in Notch signalling-deficient ES cell differentiations. In conclusion, this work demonstrates that covalent immobilisation of antibody arrays to the PIII-PC surface provides faithful cell surface antigen data in a cost- and labour-efficient manner. This may be used to facilitate high throughput identification and standardisation of more precise marker profiles during stem cell differentiation and in various genetic and disease contexts.
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, 171 77, Stockholm, Sweden, heathermain187@gmail.com.
authors.forename: Heather
authors.initials: H
authors.lastname: Main
authors.forename: Jelena
authors.initials: J
authors.lastname: Radenkovic
authors.forename: Elena
authors.initials: E
authors.lastname: Kosobrodova
authors.forename: David
authors.initials: D
authors.lastname: McKenzie
authors.forename: Marcela
authors.initials: M
authors.lastname: Bilek
authors.forename: Urban
authors.initials: U
authors.lastname: Lendahl
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology; D000954:Antigens, Surface / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D027261:Fusion Regulatory Protein-1 / Q000378:metabolism; D039482:Integrin alpha5 / Q000378:metabolism; D007477:Ions / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011076:Polycarboxylate Cement / Q000737:chemistry*; D040081:Protein Array Analysis; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D060245:Tetraspanin 29 / Q000378:metabolism
chemical_list: D000906:Antibodies; D055910:Antibodies, Immobilized; D000954:Antigens, Surface; D027261:Fusion Regulatory Protein-1; D039482:Integrin alpha5; D007477:Ions; D011076:Polycarboxylate Cement; D060245:Tetraspanin 29; C005506:polycarbonate; D019009:Proto-Oncogene Proteins c-kit