Figure 1
Figure 1. Preparation of enriched populations of CFU-E, Pro-Es, Int-Es, and Late-Es. (A) CD36, CD71 (transferrin receptor 1) and CD235a expression determined by flow cytometry of peripheral blood mononucleocyte-derived erythroblasts. Cells were stained for 15 minutes at 4°C in 2% bovine serum albumin in HBSS and washed twice. CFU-E are CD36+, CD71+, and CD235a−/low. CD235a expression increases until the intermediate erythroblast stage, when it remains high and CD71 expression begins to decrease. Stained cells were sorted on a MoFlo cell sorter (Beckman Coulter) to obtain erythroblast populations enriched for the appropriate stage of development. Our cells were tightly gated to ensure discrete populations of cells similar in maturity and lineage. Sort gates were defined by first setting a gate on the fluorescent expression profile of the population of interest, either CD71 versus CD36 (i) and/or CD71 versus CD235a (ii-v). These gates were then applied to the forward scatter versus side scatter dot plot, and a second gate was applied to exclude debris and also to define a population of cells showing similar size. The mean percentages of sorted cells collected in the total erythroid gates were 14%, 24%, 20%, and 50% for cells sorted on days 4, 5 to 6, 8 to 11, and 13 to 15, respectively. Sorted CFU-E, Pro-Es, Int-Es, and Late-Es represented an average of 28%, 29%, 37%, and 45% of erythroid cells present, respectively. (B) Cytospins of cells stained with May-Grünwald-Giemsa are shown to illustrate the morphologic changes as cultured erythroblasts mature from large CFU-E with a large, smooth nucleus at day 4, through the Pro-E and basophilic Int-E stages as the chromatin becomes more condensed, accompanied by a reduction in both cell size and the nucleus/cell size ratio, to the small Late-Es when the cytoplasm turns from blue to orange reflecting the production of hemoglobin and the nuclei become pyknotic. From all hybridized sorts, we have counted at least 250 cells per sample and find an average of 99.3% purity for our populations, with the contaminants being erythroid cells of slightly increased or decreased maturity rather than nonerythroid cells. Representative plots and cytospins viewed at 20× original magnification are shown.

Preparation of enriched populations of CFU-E, Pro-Es, Int-Es, and Late-Es. (A) CD36, CD71 (transferrin receptor 1) and CD235a expression determined by flow cytometry of peripheral blood mononucleocyte-derived erythroblasts. Cells were stained for 15 minutes at 4°C in 2% bovine serum albumin in HBSS and washed twice. CFU-E are CD36+, CD71+, and CD235a−/low. CD235a expression increases until the intermediate erythroblast stage, when it remains high and CD71 expression begins to decrease. Stained cells were sorted on a MoFlo cell sorter (Beckman Coulter) to obtain erythroblast populations enriched for the appropriate stage of development. Our cells were tightly gated to ensure discrete populations of cells similar in maturity and lineage. Sort gates were defined by first setting a gate on the fluorescent expression profile of the population of interest, either CD71 versus CD36 (i) and/or CD71 versus CD235a (ii-v). These gates were then applied to the forward scatter versus side scatter dot plot, and a second gate was applied to exclude debris and also to define a population of cells showing similar size. The mean percentages of sorted cells collected in the total erythroid gates were 14%, 24%, 20%, and 50% for cells sorted on days 4, 5 to 6, 8 to 11, and 13 to 15, respectively. Sorted CFU-E, Pro-Es, Int-Es, and Late-Es represented an average of 28%, 29%, 37%, and 45% of erythroid cells present, respectively. (B) Cytospins of cells stained with May-Grünwald-Giemsa are shown to illustrate the morphologic changes as cultured erythroblasts mature from large CFU-E with a large, smooth nucleus at day 4, through the Pro-E and basophilic Int-E stages as the chromatin becomes more condensed, accompanied by a reduction in both cell size and the nucleus/cell size ratio, to the small Late-Es when the cytoplasm turns from blue to orange reflecting the production of hemoglobin and the nuclei become pyknotic. From all hybridized sorts, we have counted at least 250 cells per sample and find an average of 99.3% purity for our populations, with the contaminants being erythroid cells of slightly increased or decreased maturity rather than nonerythroid cells. Representative plots and cytospins viewed at 20× original magnification are shown.

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