Genetix's unique ClonePix FL technology uses our ability to grow mammalian cells into clonal colonies suspended in semisolid media, and image these colonies using fluorescence assays and markers. Here we describe a new application for this technology in the isolation of clonal cell populations based on their expression of cell surface proteins. The ability to select cells based on surface protein expression is of great use in establishing cell lines expressing receptor(s) of interest for downstream phenotyping or genotyping, for use in basic discovery research, or as cell lines for cell-based assays and screening. Historically such separation of cells has been achieved using fluorescence-activated flow cytometry to sort cells based on protein expression. Using our fluorescence imaging technology, we have developed a high-powered alternative to flow cytometry that not only allows users to select the highest expressers for a particular receptor, or combination of receptors or other cell surface proteins, but also yields monoclonal populations of cells. For this application of the ClonePix FL technology, single cells are plated at a density of 300-1,000 cells/ml (depending on the cell line) in Genetix CloneMatrix-based semisolid medium (using the appropriate liquid medium base for each cell line under investigation) into black-walled Genetix PetriWell-6 plates and are allowed to grow into colonies of 50-100 cells per colony at 37 °C (7-14 d, depending on the cell line). When the colonies reach this optimal size, a fluorescent antibody to the receptor of interest is sprayed onto the semisolid medium using Genetix Atomiser bottles to achieve an even covering of antibody solution over the well. Subsequent incubation for 24-48 h allows the antibody to diffuse through the medium and bind to the colonies expressing the receptors of interest. Colonies of cells are then imaged by ClonePix FL using fluorescence and analyzed using ClonePix FL's dedicated software. The selected fluorescent colonies are automatically picked from the semisolid medium and dispensed into the wells of a 96-well destination plate. In the following proof-ofprinciple studies, the picked clonal populations of cells were analyzed after picking to confirm good growth and viability after the picking process. Selection of endogenously expressed surface proteinsWe have optimized our semisolid media to allow growth of a variety of cells into suspended clonal colonies of 50-100 cells in less than 14 d. This allows us to select cells from a mixed or heterogeneous population for their expression of endogenous cell surface proteins, using fluorescently conjugated antibody to the protein of interest ( Fig. 1).
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