Redox state analysis of definitive erythroblasts derived from ES cells.
Redox state of cells was assayed as described in “Materials and methods,” using DCFHDA as a fluorogenic substrate, but in the absence of H2O2, and cellular fluorescence intensity was examined by flow cytometry. (A) The wild-type definitive erythroblasts. (B) Alas2-null definitive erythroblasts. (C) Mouse bone marrow cells. TER119 fluorescence is shown on the ordinate (y-axis), while DCFHDA-mediated fluorescence is shown on the abscissa (x-axis). Note in the bone marrow TER119+ cells (C), cell population at the upper left exhibited a significantly lower level of DCFHDA fluorescence than the wild-type (A) andAlas2-null definitive erythroblasts (B). (D) Histogram of DCFHDA-mediated fluorescence: the wild-type definitive erythroblasts (filled curve); Alas2-null definitive erythroblasts (open curve). The open curve was shifted more to the right than the filled curve, suggesting that Alas2-null definitive erythroblasts are more peroxidized than the wild-type definitive erythroblasts.
