Strategy for flow cytometric analysis of normal bone marrow (NBM) and chronic myeloid leukaemia (CML) samples. Mononuclear cells were stained with CD34+, lineage marker, and Thy antibodies as described in “Materials and Methods.” Gates were set to sort live (propidium iodide excluding), lineage-negative cells as shown (A and B). Further gating, as shown in (C) was applied to define the CD34+Lin-Thy+ and CD34+Lin-Thy-populations revealed by staining with CD34 allophycocyanin (APC) and Thy phycoerythrin (PE). Using this four-parameter approach, CD34+Lin-Thy-(D) and CD34+Lin-Thy+ (E) populations were sorted to >90% purity. The sorted cells were then stained with rhodamine-123 (Rh-123) and Hoechst 33342 to simultaneously analyze the cell cycle and Rh-123 retention profiles of the sorted populations (F) and (G). The quadrant marker separates high levels of rhodamine-123 (Rh-123hi) from low levels of rhodamine-123 (Rh-123lo) cells and cycling cells from non cycling cells. A sample of normal bone marrow is depicted throughout this figure; cells from CML patients were processed using an identical strategy.