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. 2004 Nov 22;167(4):723–734. doi: 10.1083/jcb.200405144

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Copyright © 2004, The Rockefeller University Press

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Figure 3. — S18-induced apoptosis eliminates Oct-4(+)/PAR-4(+) pluripotent stem cells and enriches for nestin(+) NPs. (A and B) MACS sorting of apoptotic EBCs. Mouse EBCs from untreated (A) or S18-treated (80 μM; B) EBs were incubated with Annexin V–conjugated magnetic beads and fractionated using MACS. Flow through cells (Annexin V(−)) and retained cells (Annexin V(+)) were precipitated on lysine-coated coverslips, incubated with FLICA substrate, and after fixation, immunostained for the expression of PAR-4 (Cy3, red) and Oct-4 (Cy5, green). FLICA-negative and -positive cells are labeled with (−) or (+), respectively. Arrows indicate Oct-4(+)/PAR-4(+) cells. Asterisks label cells in the Annexin V(+) fraction that recovered from the initial phase of apoptosis. (C) Radial expansion and neural differentiation of mouse EBs stained for the expression of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, blue). Arrow points at cell showing coexpression and subcellular segregation of nestin and PAR-4. (D) Mouse EBs incubated overnight with 80 μM of S18 were stained for apoptotic cells (TUNEL or Annexin V, FITC, green) and nestin (Cy3, red).