The frequency of 6-thioguanine-resistant human peripheral blood lymphocytes as determined by flow cytometry and by clonal propagation

Abstract

Human peripheral blood lymphocytes were selected for 6-thioguanine resistance in short-term cultures. Resistant cells, defined as cells capable of incorporating tritiated thymidine under the selective conditions, were flow-cytometrically differentiated with respect to their DNA content. This was carried out by sorting at two stages of the cell cycle, before and after mid-S-stage, yielding frequencies of resistant cells in the range of 10(-4) and 10(-5), respectively. Observed frequencies for cells from the whole cell cycle spectrum and for cells cultured according to the long-term protocol, the clonal assay, were in the range of 10(-4) and 10(-5), respectively. Our interpretation of these results is that the over-representation of tritiated thymidine-labelled cells occurring before mid-S-stage after short-term culture reflects less resistant cells or phenocopies which are probably eliminated during long-term culture with the clonal assay, hence leading to a decreased frequency of 6-thioguanine-resistant cells. We conclude, therefore, that short-term culture in combination with flow cytometric sorting after mid-S-stage in the cell cycle can be used as an alternative to the clonal assay for the determination of fully 6-thioguanine-resistant human peripheral lymphocytes.