Erythroblastic islands from the spleen of PHZ-treated mice. (A) Cells from PHZ-injected C57BL/6J mouse spleen were stained with SYTO16 and APC-labeled anti-Ter119, and analyzed on an FACSCanto II. The staining profiles for SYTO16 and Ter119 in SytoxBlue-negative cells are shown. (B) Erythroblastic islands were stained with DAPI and Alexa488-conjugated phalloidin, and observed by FV1000D confocal microscopy with an objective lens magnification of ×60. Merged images of cells stained with DAPI (blue) and phalloidin (green) are shown. The left and right panels show images focusing on erythroblasts and central macrophages, respectively. Scale bar, 20 μm. (C) Cells in the erythroblastic islands were stained with SYTO16 and analyzed by FACS. The FSC-SSC (left panel), and the SYTO16-staining (middle panel) profiles of the SytoxBlue-negative population are shown. The SYTO16-negative population represents reticulocytes. The FSC-SSC profile of the SYTO16-positive cell population is shown on the right, in which the FSC^low and FSC^high populations represent pyrenocytes and erythroblasts, respectively. The percentage of cells in each fraction was calculated. Experiments were performed 3 times with different mice, and the average values are shown with standard deviation (vertical bars). (D) The indicated sorted cell population was cytospun and stained with Wright-Giemsa, and observed by BioRevo fluorescence microscope with objective lens magnification of ×100. (E) The erythroblastic islands were cultured overnight, and the cells in the entire culture were stained with SYTO16. The FSC-SSC- and SYTO16-staining profiles, and the percentage of each cell population are shown as described above (C).