American Society of Hematology

Figure 1

$Figure 1. Enrichment of BFU-E and CFU-E cells by flow cytometric cell sorting. (A) Day 14.5-15.5 mouse fetal liver cells stained with biotin-conjugated antibodies against murine Ter119, B220, Mac-1, CD3, Gr-1, CD32/CD16, Sca-1, CD41, and CD34 were first depleted by magnetic beads (supplemental Table 1). The enriched negative fraction (FLEP) was then stained with streptavidin-phycoerythrin (PE), CD71 fluorescein isothiocyanate (FITC), and CD117 allophycocyanin (APC) antibodies, and the phycoerythrin-negative cells were sorted by FACS into 2 fractions, called BFU-E and CFU-E. The BFU-E fraction is the 10% lowest CD71-expressing part of the Kit (CD117)+ fraction, which has a CD71 signal intensity that is similar to or slightly higher than that of unstained cells. The CFU-E fraction is the 20% highest CD71-expressing part of the Kit+ fraction. Eighty-six percent of colonies formed by sorted BFU-E cells were BFU-E colonies, 11% CFU-E colonies, and 3% other myeloid colonies. Thus, the purity of these sorted BFU-E cells is ∼ 86%. The purity of CFU-E cells is slightly higher because they form 97% CFU-E colonies, 3% late-BFU-E colonies, and no other myeloid colonies (supplemental Table 2). The same FACS setup was later used to separate BFU-E and CFU-Es with higher purities with the use o f CD24a and a combination of CD24a and CD71 (supplemental Table 2). (B) Micrographs of sorted CFU-E (Kit+ CD7120%high) cells and BFU-E (Kit+ CD710%low) cells stained with May-Grünewald-Giemsa. BFU-E cells have a high nuclear/cytoplasmic ratio and very fine nuclear chromatin. CFU-E cells are larger than BFU-E cells with a lower nuclear/cytoplasmic ratio and more regions of heterochromatin. CFU-E cells have multiple large, well-defined nucleoli (red arrows) in the nuclei. The CFU-E cytoplasm is very basophilic and sometimes bulges out from the cell. The morphology and relatively smaller size of BFU-E cells compared with CFU-E cells agree with previous studies on less pure populations of CFU-E and BFU-E cells.11,12 FS indicates forward scatter; SS, side scatter.$

Enrichment of BFU-E and CFU-E cells by flow cytometric cell sorting. (A) Day 14.5-15.5 mouse fetal liver cells stained with biotin-conjugated antibodies against murine Ter119, B220, Mac-1, CD3, Gr-1, CD32/CD16, Sca-1, CD41, and CD34 were first depleted by magnetic beads (supplemental Table 1). The enriched negative fraction (FLEP) was then stained with streptavidin-phycoerythrin (PE), CD71 fluorescein isothiocyanate (FITC), and CD117 allophycocyanin (APC) antibodies, and the phycoerythrin-negative cells were sorted by FACS into 2 fractions, called BFU-E and CFU-E. The BFU-E fraction is the 10% lowest CD71-expressing part of the Kit (CD117)⁺ fraction, which has a CD71 signal intensity that is similar to or slightly higher than that of unstained cells. The CFU-E fraction is the 20% highest CD71-expressing part of the Kit⁺ fraction. Eighty-six percent of colonies formed by sorted BFU-E cells were BFU-E colonies, 11% CFU-E colonies, and 3% other myeloid colonies. Thus, the purity of these sorted BFU-E cells is ∼ 86%. The purity of CFU-E cells is slightly higher because they form 97% CFU-E colonies, 3% late-BFU-E colonies, and no other myeloid colonies (supplemental Table 2). The same FACS setup was later used to separate BFU-E and CFU-Es with higher purities with the use o f CD24a and a combination of CD24a and CD71 (supplemental Table 2). (B) Micrographs of sorted CFU-E (Kit⁺ CD71^20%high) cells and BFU-E (Kit⁺ CD71^0%low) cells stained with May-Grünewald-Giemsa. BFU-E cells have a high nuclear/cytoplasmic ratio and very fine nuclear chromatin. CFU-E cells are larger than BFU-E cells with a lower nuclear/cytoplasmic ratio and more regions of heterochromatin. CFU-E cells have multiple large, well-defined nucleoli (red arrows) in the nuclei. The CFU-E cytoplasm is very basophilic and sometimes bulges out from the cell. The morphology and relatively smaller size of BFU-E cells compared with CFU-E cells agree with previous studies on less pure populations of CFU-E and BFU-E cells.^11,12 FS indicates forward scatter; SS, side scatter.

This Feature Is Available To Subscribers Only