Functionalization of Micropallet Arrays for Multichannel Immunofluorescent Imaging of Complex Cellular Mixtures

The micropallet array is a photoresist-based platform that provides a means to isolate and recover single adherent cells on individual pedestals, termed “micropallets”. We present a strategy to functionalize the micropallet array by incorporating multichannel immunofluorescent confocal imaging to identify cellular subsets of defined surface phenotype within complex heterogeneous mixtures, such as would be obtained from a tissue biopsy. We present the capacity to interrogate the expression pattern of up to 5 cell surface molecules. As proof of principle, we have demonstrated the capacity to detect the panel of surface molecules (Epithelial Surface Antigen (ESA), CD24, CD44, CD133, and CD309) necessary to identify unique cellular subsets within human mammary tumors, e.g. putative cancer stem cells, endothelial progenitor cells, and bulk malignant epithelium. Appropriate selection of fluorophores is critical to this capacity; monoclonal antibodies were procured directly conjugated to Brilliant Violet (BV) fluorophores (CD24-BV421 and CD44-BV605), while Alexa Fluor (AF) conjugates (CD309-AF488, ESAAF546, and CD133-AF647) were prepared using established conjugation chemistries for monoclonal antibodies not available directly conjugated to the desired fluorophores. Selected cell lines that collectively express the panel of cell surface molecules (MCF7, D283 Med, and transfected HEK 293T cells transiently expressing CD309), were applied to the micropallet array, stained with the antibody-fluorophore panel, and imaged using laser scanning confocal microscopy. The ability to interrogate the expression pattern of several cell surface molecules provides a critical addition to the existing micropallet array platform, in that it enables the identification, enumeration, and with the previously demonstrated capacity to release and retrieve micropallets carrying single adherent cells, the study of single cells of defined surface phenotype isolated from a complex heterogeneous population. This new capacity functionalizes the micropallet array platform technology to be broadly applied to the study of complex cellular systems or tissues.