Flow cytometric measurement of rRNA levels detected by fluorescent in situ hybridization in differentiating K-562 cells.
The flow cytometric detection of fluorescent in situ hybridization (FISH) performed on intact cells in suspension is a recently described method (Bauman et al. 1989). We studied the application of this method for monitoring cellular differentiation. The amount of rRNA which is taken for a good indicator of growth in size, the rate of protein synthesis and the G0 G1 transition was followed by FISH. For this purpose biotinylated single stranded RNA probes obtained by transcription from a 2.1 kb BglII-EcoRI fragment of the human 28S ribosomal RNA gene subcloned into plasmid pGEM2 were used. K-562 leukaemic cells, used as targets, were induced to differentiate by dimethyl sulfoxide, phorbol myristate acetate and hemin. In the last two cases the cell cycle analysis, growth kinetics, cellular morphology and immunophenotyping indicated differentiation into monocytic and erythroid direction, respectively. The differentiation was accompanied by a rapid increase followed by a decrease to the base level of rRNA. This was not observed in the uninduced exponentially growing control cells. Based on our results, we propose that the FC-FISH detection of the rRNA level is a valuable method to distinguish between cell subpopulations. We propose that using other probes, FC-FISH will become useful to monitor different cellular processes.
Department of Pathology, University Medical School of Pécs, Hungary.
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Pajor
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JG
Bauman
D001710:Biotin / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D016130:Immunophenotyping; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D015347:RNA Probes / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000032:analysis*; D014407:Tumor Cells, Cultured
D015347:RNA Probes; D012339:RNA, Ribosomal, 28S; D001710:Biotin