Sorting of Rare Cells
Positive selection on the basis of a particular phenotype is the most efficient way for the isolation of cells occurring at frequencies of below 1% in a given population. As one of the most powerful methods for positive selection, fluorescence-activated cell sorting (FACS) has been used for the enrichment and isolation of rare cells ever since its introduction (Assenmacher et al., 1990). Examples include immunoglobulin switch variants (Radbruch et al., 1980, Dangl et al., 1982), MHC mutants (Holtkamp et al., 1981, Weichel et al., 1985), and transfectants (Kavathas et al., 1984, Hombach et al., 1988). Such cells occur at frequencies of less than 10−2down to 10−8 in some cases. In general, rare cells were isolated in several rounds of cell sorting and expanding the enriched cells between the sorts in vitro; thereby gradually increasing the frequency of variants until isolation of rare cells and cloning became possible. Under optimal conditions, enrichment rates of up to 103 per sort could be achieved, but the number of sorted cells was usually too low to allow immediate resorting for purification of the rare cells. Naturally, such experiments can only be performed with transformed cells, adapted to continuous growth in culture. Although this approach still is the method of choice in many experimental situations, additional parameters are available in flow cytometry today and new methods for fast and efficient enrichment of cells prior to FAGS sorting have been introduced, substantially facilitating the isolation of rare cells. Here we describe a combined protocol for preselection of rare cells by magnetic cell sorting followed by fluorescence activated enrichment or even isolation of the same cells in one experiment. However, the FACS sorting part of this procedure also applies for enrichment problems where MACS enrichment is not possible, for instance the isolation of rare cells expressing green fluorescent protein as a marker.
KeywordsFiltration Convection Albumin Sedimentation Syringe
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