Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures.
Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19(+) Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.
Department of Life and Medical Sciences, University of Bonn, Bonn, Germany.
Günter
G
Mayer
Marie-Sophie L
MS
Ahmed
Andreas
A
Dolf
Elmar
E
Endl
Percy A
PA
Knolle
Michael
M
Famulok
D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D015723:Gene Library; D052156:SELEX Aptamer Technique / Q000379:methods*; D014407:Tumor Cells, Cultured