Identification and isolation of purified ALDH^hiLin^- and ALDH^loLin^- cell populations. Representative flow cytometric analysis of ALDH activity on lineage-depleted (Lin^-) UCB cells. (A) Human UCB Lin^- cells were selected according to forward scatter (FSC) and side scatter (SSC) properties using the gated region R1 to remove residual platelet and red cell contamination. (B) Lin^- cells incubated with Aldefluor substrate and the specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), were used to establish baseline fluorescence of these cells and to define the ALDH^hi region (R3) as less than 0.5% of total events. (C) Incubation of Lin^- cells with Aldefluor substrate in the absence of inhibitor induced a shift in FL1 fluorescence defining the ALDH^lo (R2 = 31.5% ± 1.8%) and ALDH^hi (R3 = 50.1% ± 2.4%) purified populations selected on the basis of intracellular ALDH conversion of Aldefluor substrate. (D) Lin^- cells incubated with Aldefluor substrate were reanalyzed by using FL1 fluorescence versus SSC to ensure that mononuclear cells with equivalent side scatter properties were selected by sorting. These data represent the mean ± SEM on experiments performed with Lin^- cells isolated from UCB samples from 9 different donors (n = 9).