Figure 2.
Dissection of myeloid-erythroid progenitor activity in the CD45+ fraction from EB cultures. (A) Staining of EB cultures maintained in the 6-cytokine cocktail with antibodies against CD14 and CD11b evidenced at least 3 discrete population. These were purified using FACS (reanalysis of purity shown) and plated at equal numbers into methylcellulose supplemented with myelo-erythroid lineage-inducing cytokines. (B) Methylcellulose colonies were enumerated at day 10 of culture. (C) Bright-field image of the most primitive colony type, CFU-GEM, from the CD14CD11b DN sort. (D) Individual colonies from the methylcellulose cultures of CD11bCD14 DN sorted cells were cytospun onto glass slides and stained with Wright-Giemsa kit HEMA 3 (Fisher Scientific) (7 colonies shown) and (E) evaluated by flow cytometry for the expression of CD11b, CD11c, CD14, and CD235 (2 representative colonies shown). BFU-E, burst-forming unit-erythroid; CFU- colony forming unit; E, erythrocyte; G, granulocyte; GEM, granulocyte, erythrocyte, and monocyte; GM, granulocyte and monocyte; M, monocyte.

Dissection of myeloid-erythroid progenitor activity in the CD45+ fraction from EB cultures. (A) Staining of EB cultures maintained in the 6-cytokine cocktail with antibodies against CD14 and CD11b evidenced at least 3 discrete population. These were purified using FACS (reanalysis of purity shown) and plated at equal numbers into methylcellulose supplemented with myelo-erythroid lineage-inducing cytokines. (B) Methylcellulose colonies were enumerated at day 10 of culture. (C) Bright-field image of the most primitive colony type, CFU-GEM, from the CD14CD11b DN sort. (D) Individual colonies from the methylcellulose cultures of CD11bCD14 DN sorted cells were cytospun onto glass slides and stained with Wright-Giemsa kit HEMA 3 (Fisher Scientific) (7 colonies shown) and (E) evaluated by flow cytometry for the expression of CD11b, CD11c, CD14, and CD235 (2 representative colonies shown). BFU-E, burst-forming unit-erythroid; CFU- colony forming unit; E, erythrocyte; G, granulocyte; GEM, granulocyte, erythrocyte, and monocyte; GM, granulocyte and monocyte; M, monocyte.

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