Binding of erythroblasts to Vcam1-expressing macrophages. (A) Cells in the erythroblastic islands were suspended in PBS/BSA/EDTA, and stained with biotinylated hamster anti-F4/80, followed by staining with SytoxBlue, APC-labeled anti-hamster IgG, and PE-labeled anti-Vcam1, and analyzed on an FACSCanto II. Macrophages emit autofluorescence in the Pacific blue channel (blue laser; excitation 405, emission 450/50).18 Thus, the SytoxBluelow population (upper panel) was analyzed for F4/80 and Vcam1 expression with or without primary antibodies (lower panels). (B) Vcam1-dependent binding of erythroblasts to central macrophages. Cells in erythroblastic islands were suspended in PBS/BSA/EDTA and stained with PE-anti-Ter119. The cells were then incubated in HBS/BSA/EDTA, or in HBS/BSA/CaMg in the presence of 10 μg/mL control IgG or in the indicated amount of anti-Vcam1 at RT for 30 minutes, and analyzed on an FACSCanto II. The Ter119-staining profiles associated with the SytoxBluelow macrophage population (upper panel) are shown. (C) The erythroblastic islands were cultured overnight, washed with HBS/CaMg, and the cells in the islands were stained with SYTO16. The FSC-SSC- and SYTO16-staining profiles, and the percentage of each cell population are shown. (D) BaF3 and BaF3/Vcam1 cells were incubated with CellTracker-labeled erythroblasts, reticulocytes, or pyrenocytes at RT for 30 minutes in HBS/BSA/CaMg, and analyzed on an FACSCanto II (supplemental Figure 2, available on the Blood Web site). The numbers indicate the percentage of CellTracker-positive cells. Experiments were performed in triplicate, and the average values are shown with standard deviation. (E) Cells from the PHZ-treated mouse spleen were incubated for 5 hours at 37°C. The cells were then incubated with 10 nM LDV-FITC for 30 minutes at RT in HBS/BSA/EDTA, HBS/BSA/CaMg, or HBS/BSA/CaMgMn, stained with Draq5 and SytoxBlue, and analyzed on an FACSCanto II. The FITC mean fluorescence intensity (MFI) in the SytoxBlue− fraction was determined for the Draq+FSChigh (erythroblasts), Draq+FSClow (pyrenocytes), and Draq− (reticulocytes) populations. Experiments were performed in triplicate, and the average values were plotted with standard deviation (vertical bars). All experiments in Figure 2 were carried out several times with erythroblastic islands prepared from different mice, and representative results are shown.