Simultaneous use of Rhodamine 123, phycoerythrin, Texas red, and allophycocyanin for the isolation of human hematopoietic progenitor cells

Abstract

The supravital, mitochondrial specific dye Rhodamine 123 (R123) was used in conjunction with three monoclonal antibodies to isolate a population of human bone marrow (BM) cells enriched for hematopoietic progenitor cells. BM cells stained with phycoerythrin-HLA-DR, Texas red-CD34, allophycocyanin-CD15, and R123 were fractionated using four-color immunofluorescence cell sorting. Cells expressing CD34 but not HLA-DR and CD15 (CD34⁺ HLA-DR⁻ CD15⁻) were subdivided according to their reactivity with R123 into quiescent, R123 dull (R⁺) or cycling, R123 bright (R⁺⁺) subpopulations. Morphological analysis and hematopoietic progenitor cell assays indicated that CD34⁺ HLA-DR⁻ CD15⁻ R⁺ cells contained larger numbers of blast cells and colony forming units than CD34⁺ HLA-DR⁻ CD15⁻ R⁺⁺ cells. The flow cytometer settings used to accommodate the detection of the R123 fluorescence in combination with that of three other fluorochromes are described.