Flow cytometry has become an integral part of modern cell biology. Flow cytometers utilise a range of different lasers to excite fluorophores bound to cell surface biomarkers, as the cells move past in a single-file flow. Both the scattered light from the excitation source and emitted light from the fluorophores are used to count (and quantify) the size, number and fluorescence intensity (and hence biomarker expression level) of each individual cell. This data can then be used to determine the expression profile of a cell population on a cell-by-cell basis. The use of multiple lasers, affinity ligands and attached fluorophores allows simultaneous analysis of several markers expressed on thousands of cells per second. This makes it a rapid and quantitative method for analysis and purification of cells in suspension.
The analysed cells may also be separated based on these fluorescent properties using Fluorescence-Activated Cell Sorting (FACS). Here, the user defines the parameters upon which, cells will be sorted. The instrument then imparts an electrical charge on each cell so that they may be separated using electromagnets.
As with any analytical technique; Flow Cytometry and FACS are only as good as the affinity ligand used. For example, if the fluorescently labelled antibody is not specific or the chosen biomarker is not unique to the cells of interest, false positive results will be seen. Antibody over-labelling may also lead to a significant loss of function, if the non-specific conjugation of a fluorophore interferes with the antigen binding domain. Antibody stability can also be a significant factor in producing reliable data.
Flow cytometry data shows autofluorescence for both cell lines incubated without aptamer (blue traces). An individual aptamer (FITC labelled) shows concentration dependent binding to its positive target cell line (left) but not the negative target cells (right).
Cell-based aptamer selection avoids many of the issues faced by antibodies used in Flow Cytometry and FACS.
- The aptamers can be isolated directly using intact cells / tissues – the antigen is therefore recognised in its native context (embedded in the cell wall)
- The nature of the target protein does not need to be known – aptamer selection can be ‘hypothesis-free’ and target all cell surface proteins simultaneously.
- Aptamers are produced by solid phase synthesis methodologies – labelling steps (or appropriate function groups) can be incorporated during synthesis to enable reliable site-specific labelling