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. 2010 Jan 5;5(1):e8581. doi: 10.1371/journal.pone.0008581

Figure 6. Chk1+/− mice show a disruption in enucleation of erythrocytes in the bone marrow.

Figure 6

(A) Representative flow cytometry plot of enucleation assay used. Syto 16 is cell permeable, whereas Sytox Blue is cell impermeable, thus the two dyes in tandem allow the separation of enucleated and nucleated erythrocytes [6]. Low FSC Ter-119+ cells or high FSC Ter-119+ were examined for the percentage of nucleated or enucleated cells. Low FSC (small) cells are the more mature erythrocytes, while high FSC (large) cells are the more immature erythrocytes. (B) The P1 gated cells, which should almost entirely consist of RBCs, have fewer properly enucleated erythrocytes with an increase in cells having their nuclei in Chk1+/− whole bone marrow. The P2 gated cells, the majority of which should be immature erythrocytes with their nuclei still intact show a decrease in nucleated cells with a concurrent increase in enucleated cells in the Chk1+/− whole bone marrow. (C) Progenitors from stages I–IV were sorted from WT and Chk1+/− mice and immunostained with Alexa-flour 594 Phalloidin(red) and DAPI(blue). The WT erythroid progenitors from stages I–IV show dynamics of actin filaments during CAR in erythropoiesis. WT erythroid progenitors in Stages III and IV display formation of CAR (yellow arrows). Aberrant CAR formation (yellow arrows) is observed in stages III and IV Chk1+/− erythroid progenitors at 60× magnification and images scaled at 20 µm.