Fig. 2.
Fig. 2. Effect of immobilized Deltaext-myc on differentiation of monocytes cultured with either GM-CSF or GM-CSF and IL-4 with or without TNF-α. / (A) Fluorescence histograms of peripheral monocytes and cells cultured with GM-CSF or GM-CSF and IL-4 in the presence of 1 μg/mL Deltaext-myc or control medium. Tissue culture wells were coated with anti-myc antibody, 9E10 F(ab′)2, to attach myc-containing Deltaext-myc to the plastic surface. The x-axis represents log fluorescence intensity and the y-axis represents cell number. The shaded histograms represent staining with antibodies against CD14 (Leu-M3), CD64, CD1a, HLA-DR, CD80, CD86, and CD40, and open histograms represent staining with an isotype-matched control antibody of irrelevant specificity. Data are representative of 5 experiments. (B) Effect of Deltaext-myc on TNF-α–induced maturation of immature dendritic cells. Immature dendritic cells derived from monocytes cultured for 5 days with GM-CSF and IL-4 (top panel). Cells were then incubated with GM-CSF, IL-4, TNF-α, and either 1 μg/mL Deltaext-myc or control medium for 2 days (bottom 2 panels). Tissue culture wells were coated with anti-myc antibody, 9E10 F(ab′)2. The shaded histograms represent staining with designated antibodies and open histograms represent staining with an isotype-matched control antibody. One representative experiment of 3 is shown. (C) MLR-stimulatory capacity of cultured cells. In left panel, stimulator cells were prepared from monocytes cultured for 6 days with GM-CSF and 1 μg/mL Deltaext-myc(●), GM-CSF and control medium (○), or GM-CSF and IL-4 (▴). In the right panel, stimulator cells were prepared from monocytes cultured for 7 days with GM-CSF and IL-4 in the presence of 1 μg/mL Deltaext-myc (●) or control medium (○), or from monocyte-derived immature dendritic cells cultured for 2 days with GM-CSF, IL-4, and TNF-α in the presence of 1 μg/mL Deltaext-myc (▴) or control medium (▵). All wells were coated with anti-myc antibody, 9E10 F(ab′)2. After irradiation, increasing numbers of stimulator cells were cocultured with PBMC (5 × 104) for 5 days, and3H-thymidine uptake was assessed. Values are the mean ± SD obtained from triplicate cultures. Data are representative of 3 experiments.

Effect of immobilized Deltaext-myc on differentiation of monocytes cultured with either GM-CSF or GM-CSF and IL-4 with or without TNF-α.

(A) Fluorescence histograms of peripheral monocytes and cells cultured with GM-CSF or GM-CSF and IL-4 in the presence of 1 μg/mL Deltaext-myc or control medium. Tissue culture wells were coated with anti-myc antibody, 9E10 F(ab′)2, to attach myc-containing Deltaext-myc to the plastic surface. The x-axis represents log fluorescence intensity and the y-axis represents cell number. The shaded histograms represent staining with antibodies against CD14 (Leu-M3), CD64, CD1a, HLA-DR, CD80, CD86, and CD40, and open histograms represent staining with an isotype-matched control antibody of irrelevant specificity. Data are representative of 5 experiments. (B) Effect of Deltaext-myc on TNF-α–induced maturation of immature dendritic cells. Immature dendritic cells derived from monocytes cultured for 5 days with GM-CSF and IL-4 (top panel). Cells were then incubated with GM-CSF, IL-4, TNF-α, and either 1 μg/mL Deltaext-myc or control medium for 2 days (bottom 2 panels). Tissue culture wells were coated with anti-myc antibody, 9E10 F(ab′)2. The shaded histograms represent staining with designated antibodies and open histograms represent staining with an isotype-matched control antibody. One representative experiment of 3 is shown. (C) MLR-stimulatory capacity of cultured cells. In left panel, stimulator cells were prepared from monocytes cultured for 6 days with GM-CSF and 1 μg/mL Deltaext-myc(●), GM-CSF and control medium (○), or GM-CSF and IL-4 (▴). In the right panel, stimulator cells were prepared from monocytes cultured for 7 days with GM-CSF and IL-4 in the presence of 1 μg/mL Deltaext-myc (●) or control medium (○), or from monocyte-derived immature dendritic cells cultured for 2 days with GM-CSF, IL-4, and TNF-α in the presence of 1 μg/mL Deltaext-myc (▴) or control medium (▵). All wells were coated with anti-myc antibody, 9E10 F(ab′)2. After irradiation, increasing numbers of stimulator cells were cocultured with PBMC (5 × 104) for 5 days, and3H-thymidine uptake was assessed. Values are the mean ± SD obtained from triplicate cultures. Data are representative of 3 experiments.

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