Figure 3
Figure 3. Engulfment of pyrenocytes in erythroblastic islands. (A-D) Erythroblastic islands were cultured overnight in IMDM containing 15% FCS, 3 U/mL hEPO, and 200 μg/mL transferrin in the presence or absence of 1% methylcellulose. (A) An erythroblastic island cultured in the presence of methylcellulose is observed by FV1000D microscope with an objective lens magnification of ×60, and is shown on the left. An erythroblast, in which enucleation and engulfment of the expelled pyrenocyte were followed every 86 seconds (right panels and supplemental Video 2), is circled in yellow. Yellow arrowheads in the right panels point to enucleation of the erythroblast and engulfment of its pyrenocyte. (B) Quantification of the number of pyrenocytes engulfed during a 14-hour incubation in the presence or absence of 1% methylcellulose. The number of erythroblastic islands examined in the cultures with and without methylcellulose was 32 and 34, respectively. (C-D) After an overnight culture in 1% methylcellulose, the erythroblastic islands were washed to remove erythroblasts and reticulocytes, stained with PI, and observed by BioRevo fluorescence microscopy with an objective lens magnification of ×40 (C), or subjected to FACS analysis (D). As a control, the erythroblastic islands were depleted of erythroblasts before the overnight culture. (C) macrophages carrying dark debris can be distinguished from erythroblasts (pointed by arrowheads) that are smaller than macrophages. Scale bar, 20 μm. (D) The PI-staining profiles for the SytoxBluelow macrophages are shown. The experiments were performed 3 times, and the percentage of cells containing extra PI-positive material is shown with standard deviation. (E-H) Erythroblastic islands were cultured overnight in the presence of 1% methylcellulose, with or without the indicated concentrations of protein S. After removing erythroblasts, the adherent cells were stained with PI and observed by fluorescence microscopy (E), or analyzed on an FACSCanto II (F), as described in Figure 3C-D. The experiments were performed 3 times, and the percentage of cells containing extra PI-positive material is shown with a standard deviation (vertical bars) (G). (H) The adherent cells in erythroblastic islands cultured in the absence or presence of 15% FCS were observed by electron transmission microscope with magnification of ×2500. Yellow arrowheads indicate the engulfed pyrenocytes.

Engulfment of pyrenocytes in erythroblastic islands. (A-D) Erythroblastic islands were cultured overnight in IMDM containing 15% FCS, 3 U/mL hEPO, and 200 μg/mL transferrin in the presence or absence of 1% methylcellulose. (A) An erythroblastic island cultured in the presence of methylcellulose is observed by FV1000D microscope with an objective lens magnification of ×60, and is shown on the left. An erythroblast, in which enucleation and engulfment of the expelled pyrenocyte were followed every 86 seconds (right panels and supplemental Video 2), is circled in yellow. Yellow arrowheads in the right panels point to enucleation of the erythroblast and engulfment of its pyrenocyte. (B) Quantification of the number of pyrenocytes engulfed during a 14-hour incubation in the presence or absence of 1% methylcellulose. The number of erythroblastic islands examined in the cultures with and without methylcellulose was 32 and 34, respectively. (C-D) After an overnight culture in 1% methylcellulose, the erythroblastic islands were washed to remove erythroblasts and reticulocytes, stained with PI, and observed by BioRevo fluorescence microscopy with an objective lens magnification of ×40 (C), or subjected to FACS analysis (D). As a control, the erythroblastic islands were depleted of erythroblasts before the overnight culture. (C) macrophages carrying dark debris can be distinguished from erythroblasts (pointed by arrowheads) that are smaller than macrophages. Scale bar, 20 μm. (D) The PI-staining profiles for the SytoxBluelow macrophages are shown. The experiments were performed 3 times, and the percentage of cells containing extra PI-positive material is shown with standard deviation. (E-H) Erythroblastic islands were cultured overnight in the presence of 1% methylcellulose, with or without the indicated concentrations of protein S. After removing erythroblasts, the adherent cells were stained with PI and observed by fluorescence microscopy (E), or analyzed on an FACSCanto II (F), as described in Figure 3C-D. The experiments were performed 3 times, and the percentage of cells containing extra PI-positive material is shown with a standard deviation (vertical bars) (G). (H) The adherent cells in erythroblastic islands cultured in the absence or presence of 15% FCS were observed by electron transmission microscope with magnification of ×2500. Yellow arrowheads indicate the engulfed pyrenocytes.

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