Functional hierarchy in Li-7 cells in vitro. a) Flow cytometry of cells prepared for an Aldefluor assay and immunostained for CD13 and CD166. Upper panels: Aldefluor assay of the bulk Li-7 cell population (left: BAAA with DEAB, middle: only BAAA) and CD13 and CD166 (right). Lower panels: Aldefluor assay of gated fractions (left: CD13(+)/CD166(−), middle: CD13(−)/CD166(−), right: CD13(−)/CD166(+), showing the highest and the lowest ALDH activities in the CD13(+)/CD166(−) and CD13(−)/CD166(+) cells, respectively. b) Spheroid colony assay showed a high ability of CD13(+)/CD166(−) cells and a relatively low ability of CD13(−)/CD166(+) cells to produce colonies. c) 5-FU treatment of the subfractions showed a relatively higher sensitivity of CD13(−)/CD166(+) cells and lower sensitivity of CD13(+)/CD166(−) cells (72 hr; WST-8 assay). d) A microarray analysis showed relatively higher levels of expression of stemness-related genes in CD166(−) cells than CD166(+) cells.