Clonogenic assay combined with flow cytometric cell sorting for cell-cycle analysis of human leukemic colony-forming...

The purpose of this study is to develop a method combining clonogenic assay and flow cytometric cell sorting based on DNA content for determination of percentage distribution of human malignant clonogenic cells over cell-cycle phases. Flow cytometric cell sorting was carried out for Chinese Hamster Ovary (CHO) and human leukemic bone marrow cells under sterile conditions using a supravital dye for DNA, 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI). The fraction sorted for G0/G1 CHO cells was verified to be almost completely free of G2/M cells and partially cleared of S-phase cells. Cells sorted for G0/G1 fraction were successfully grown as colonies in culture for both CHO and human leukemic marrow cells. The plating efficiency of sorted cells was lower than that of unsorted cells, and the relative contribution of cell damage versus cell selection cannot be determined for this reduction of plating efficiency. The purity of sorted cells must still be improved, and the reason for reduced plating efficiency remains to be defined by further studies. Nonetheless this report represents initial data demonstrating the feasibility of combining two technologies: clonogenic assay and flow cytometric cell sorting based on DNA content. As such, it paves the way for a new approach to studying the cell cycle kinetics of clonogenic cell subpopulations of malignant tissues freshly obtained from patients.