Aldefluor staining is not determined by Aldh1a1 function, and HSCs cannot be distinguished from bone marrow cells based on Aldefluor staining. (A) whole bone marrow (left) and CD150⁺CD48⁻CD41⁻Sca1⁺c-kit⁺ HSCs (right; cells highlighted in orange) were (B) analyzed for Aldefluor fluorescence in the presence and absence of the ALDH inhibitor DEAB. Aldh1a1 deficiency had no effect on Aldefluor staining. (C) Mean Aldefluor fluorescence intensity minus background fluorescence in the presence of DEAB (at least 2 independent experiments per age involving a total of 3 to 5 mice per treatment). Error bars represent standard deviation. (D) Representative plots of whole bone marrow cells (black dots) and HSCs (orange dots) from (A) show SSC versus Aldefluor staining. Note that mouse HSCs did not exhibit high levels of Aldefluor staining relative to whole bone marrow. Similar results were obtained using c-kit⁺Flk-2⁻Lineage⁻Sca1⁺ HSCs (Figure S1).