American Society of Hematology

Figure 3

$Figure 3. The GATA2-deficient BMMCs are able to differentiate into functional neutrophil- and macrophage-like cells, but not into eosinophils, in the presence of myeloid cytokines. (A) The results of the Q-PCR analyses of receptors of the myeloid linage in ΔCF/ΔCF BMMCs. The PCR products amplified from ΔCF/ΔCF BMMCs on days 0, 3, 6, and 8 of the 4-OHT treatment (D0, D3, D6, and D8, respectively) are shown. N = 4. (B) Representative cytospin preparations stained with Wright-Giemsa (upper; original magnification, ×400) and phase-contrast photomicrographs (lower; original magnification, ×200) of ΔCF/ΔCF BMMCs cultured with the cytokine cocktail for 5, 10, and 15 days following the 10 days of 4-OHT treatment (D0, D5, and D10, respectively). (C) (Upper) Results of a FACS analysis for eosinophils (CD34−CD11bintCCR3+Siglec-F+) from ΔCF/ΔCF BMMCs cultured with the culture media appropriate for eosinophils containing 20 ng/mL of IL-5 (Supplemental methods) for 3 and 6 days (D3 and D6, respectively) following 10 days of 4-OHT treatment. The average percentages and SD of the gated cells are indicated. N = 3. The results of the FACS analysis of bone marrow cells from wild-type mice (BM cells) cultured for 3 days under the same culture conditions are shown in the right panel. (Lower) Cytospin preparations stained with Diff-quick from the ΔCF/ΔCF BMMCs and BM cells cultured for 6 days under the conditions described above. (D) (Top) Results of a FACS analyses of neutrophil markers on ΔCF/ΔCF BMMCs cultured under the conditions for neutrophil amplification for 6 days. (Middle) Average percentages and SD of the gated fractions (left); cytospin preparations of the isolated Ly6G+Ly6G/C+ neutrophil-like cells stained with Wright-Giemsa (right). (Bottom, left) Results of the FACS analyses of the Alexa Fluor 488-conjugated E coli bioparticles phagocytosed by the MHC class II− and Ly6G/C+ cells cultured at 4°C and 37°C. (Right) Average percentages and SD of the gated fractions. The representative data from 3 independent analyses are shown. (E) (Top) Results of the FACS analyses of macrophage markers (CD11b and F4/80) on ΔCF/ΔCF BMMCs cultured under the conditions for macrophage amplification for 6 days. (Middle) Average percentages and SD of the gated fractions (left); cytospin preparations of the isolated CD11b+F4/80+ macrophage-like cells stained with Wright-Giemsa (right). (Bottom, left) FACS histogram of microspheres phagocytosed by the F4/80+ cells cultured at 37°C. (Right) Average percentages and SD of the phagocytosed cells cultured at 4°C and 37°C. The representative data from 4 independent analyses are shown. Original magnification, ×400 for C-E.$

The GATA2-deficient BMMCs are able to differentiate into functional neutrophil- and macrophage-like cells, but not into eosinophils, in the presence of myeloid cytokines. (A) The results of the Q-PCR analyses of receptors of the myeloid linage in ΔCF/ΔCF BMMCs. The PCR products amplified from ΔCF/ΔCF BMMCs on days 0, 3, 6, and 8 of the 4-OHT treatment (D0, D3, D6, and D8, respectively) are shown. N = 4. (B) Representative cytospin preparations stained with Wright-Giemsa (upper; original magnification, ×400) and phase-contrast photomicrographs (lower; original magnification, ×200) of ΔCF/ΔCF BMMCs cultured with the cytokine cocktail for 5, 10, and 15 days following the 10 days of 4-OHT treatment (D0, D5, and D10, respectively). (C) (Upper) Results of a FACS analysis for eosinophils (CD34⁻CD11b^intCCR3⁺Siglec-F⁺) from ΔCF/ΔCF BMMCs cultured with the culture media appropriate for eosinophils containing 20 ng/mL of IL-5 (Supplemental methods) for 3 and 6 days (D3 and D6, respectively) following 10 days of 4-OHT treatment. The average percentages and SD of the gated cells are indicated. N = 3. The results of the FACS analysis of bone marrow cells from wild-type mice (BM cells) cultured for 3 days under the same culture conditions are shown in the right panel. (Lower) Cytospin preparations stained with Diff-quick from the ΔCF/ΔCF BMMCs and BM cells cultured for 6 days under the conditions described above. (D) (Top) Results of a FACS analyses of neutrophil markers on ΔCF/ΔCF BMMCs cultured under the conditions for neutrophil amplification for 6 days. (Middle) Average percentages and SD of the gated fractions (left); cytospin preparations of the isolated Ly6G⁺Ly6G/C⁺ neutrophil-like cells stained with Wright-Giemsa (right). (Bottom, left) Results of the FACS analyses of the Alexa Fluor 488-conjugated E coli bioparticles phagocytosed by the MHC class II⁻ and Ly6G/C⁺ cells cultured at 4°C and 37°C. (Right) Average percentages and SD of the gated fractions. The representative data from 3 independent analyses are shown. (E) (Top) Results of the FACS analyses of macrophage markers (CD11b and F4/80) on ΔCF/ΔCF BMMCs cultured under the conditions for macrophage amplification for 6 days. (Middle) Average percentages and SD of the gated fractions (left); cytospin preparations of the isolated CD11b⁺F4/80⁺ macrophage-like cells stained with Wright-Giemsa (right). (Bottom, left) FACS histogram of microspheres phagocytosed by the F4/80⁺ cells cultured at 37°C. (Right) Average percentages and SD of the phagocytosed cells cultured at 4°C and 37°C. The representative data from 4 independent analyses are shown. Original magnification, ×400 for C-E.

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