DNA versus RNA staining of UCB CD34⁺ cells, postsort analysis of sorted G₀ and G₁CD34⁺ cells, and reanalysis of G₀and G₁CD34⁺ cells with 7-AAD and Ki-67 staining.

(A) Fluorescence analysis of UCB CD34⁺ cells stained with Hst and PY as described in “Materials and methods.” Quiescent cells residing in G₀ (G₀CD34⁺) have 2n DNA and minimal RNA content (cells falling below the horizontal line in dot plot A), whereas those in early or late G₁ (G₁CD34⁺) are also Hst^dim but are Py^bright owing to their higher RNA content (cells above the horizontal line). According to this definition, approximately 33% of UCB CD34⁺ cells were determined to be in G₀. Sort windows to collect G₀ and G₁ cells were constructed as previously described16 for MPB and are shown in dot plot A as R1 and R2, respectively. In all sorts, at least 150 fluorescence channels separated the 2 sort windows. (B, C) Postsort analysis of sorted G₁CD34⁺ cells (dot plot B) and G₀CD34⁺ cells (dot plot C). Since PY fluorescence is lost with time, sorted cells “fall” below sort windows upon postsort analysis. (D,E) Cell-cycle status of sorted cells was confirmed by 7-AAD and Ki-67 staining. Most PY^brightcells (cells in G₁; panel D) expressed Ki-67 (86%), whereas a substantially smaller fraction (34%) of cells in G₀ (panel E) expressed low levels of Ki-67. Ki-67 and 7-AAD staining was repeated in 3 other experiments with similar results.