Fig. 3.
Fig. 3. A3D8 induces incomplete maturation and apoptosis of NB4 cells. / (A) Increased expression of myeloid differentiation antigens. Cells were treated with either 1.25 μg/mL A3D8 (Ai) or with increasing doses of this mAb (Aii). At the indicated days, cells were labeled in triplicate with FITC-conjugated mAbs directed to CD15 and CD11b, and their expression was measured by flow cytometry relative to isotypic controls. Dead cells were labeled by propidium iodide staining and excluded from the analysis. Because CD15 was expressed on all control cells, only its MFI was analyzed. Controls were cells treated with IgG1 or 2.5 μg/mL J173 anti-CD44 mAb, which was inactive. Data are mean values ± 1 SD from 3 independent experiments. ▪: control; ■: + A3D8. (B) Induction of cell death by A3D8. The percentage of dead cells was determined in triplicate, during 6 days, after addition of increasing doses of A3D8 (0.3 μg/mL to 2.5 μg/mL) using trypan blue exclusion assay. It was time- and dose-dependent. Data are mean values ± 1 SD from 3 independent experiments. (C) Apoptotic cells, 3 days following addition of 2.5 μg/mL A3D8. (Ci) Fluorescent staining with acridine orange and ethidium bromide showed nuclei condensation and fragmentation that are apoptosis-specific. Cells at an early stage of apoptosis (without loss of membrane integrity) displayed a bright green–stained nucleus. Apoptic cells that have lost membrane integrity displayed bright orange–stained nuclei as a result of taking up ethidium bromide. Magnification: × 630. (Cii) DNA strand breaks, which are apoptosis-specific, were labeled using TUNEL reaction. Their amount was evaluated by flow cytometry analysis. The proportion of labeled (apoptotic) cells was measured by flow cytometry analysis relative to negative controls labeled with dUTP without terminal deoxynucleotidyl transferase. This technique showed 39% ± 6% of apoptotic cells in the treated group versus less than 5% in controls (treated with J173). One experiment representative of 3 independent experiments is shown.

A3D8 induces incomplete maturation and apoptosis of NB4 cells.

(A) Increased expression of myeloid differentiation antigens. Cells were treated with either 1.25 μg/mL A3D8 (Ai) or with increasing doses of this mAb (Aii). At the indicated days, cells were labeled in triplicate with FITC-conjugated mAbs directed to CD15 and CD11b, and their expression was measured by flow cytometry relative to isotypic controls. Dead cells were labeled by propidium iodide staining and excluded from the analysis. Because CD15 was expressed on all control cells, only its MFI was analyzed. Controls were cells treated with IgG1 or 2.5 μg/mL J173 anti-CD44 mAb, which was inactive. Data are mean values ± 1 SD from 3 independent experiments. ▪: control; ■: + A3D8. (B) Induction of cell death by A3D8. The percentage of dead cells was determined in triplicate, during 6 days, after addition of increasing doses of A3D8 (0.3 μg/mL to 2.5 μg/mL) using trypan blue exclusion assay. It was time- and dose-dependent. Data are mean values ± 1 SD from 3 independent experiments. (C) Apoptotic cells, 3 days following addition of 2.5 μg/mL A3D8. (Ci) Fluorescent staining with acridine orange and ethidium bromide showed nuclei condensation and fragmentation that are apoptosis-specific. Cells at an early stage of apoptosis (without loss of membrane integrity) displayed a bright green–stained nucleus. Apoptic cells that have lost membrane integrity displayed bright orange–stained nuclei as a result of taking up ethidium bromide. Magnification: × 630. (Cii) DNA strand breaks, which are apoptosis-specific, were labeled using TUNEL reaction. Their amount was evaluated by flow cytometry analysis. The proportion of labeled (apoptotic) cells was measured by flow cytometry analysis relative to negative controls labeled with dUTP without terminal deoxynucleotidyl transferase. This technique showed 39% ± 6% of apoptotic cells in the treated group versus less than 5% in controls (treated with J173). One experiment representative of 3 independent experiments is shown.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal