FBZ and CHOP induced the apoptosis and necrosis of EL-4 cells. For cell death analysis, EL-4 cells (2 × 10 5 cells/mL) were treated with FBZ or CHOP. (A) After 72 h incubation, the cells were double-stained with Hoechst 33342 and PI, and the images were captured using a fluorescence microscope. Early apoptotic cells (arrows) are the cells with blue chromatin, which is highly condensed, marginated, or fragmented. Late apoptotic cells (arrow heads) indicate the cells with bright red condensed and fragmented chromatin. Necrotic cells (open arrows) are the cells with bright red or red enlarged nuclei with normal structures. Scale bar: 50 µm. (B) After 48 h incubation, annexin V/PI staining was performed. The quadrants of the dot plot indicate live cells (annexin V − /PI − ) and cells in early apoptosis (annexin V + /PI − ), late apoptosis (annexin V + /PI + ), and necrosis (annexin V − /PI + ). The graphs were generated using data from three independent experiments. Statistical significance was performed via two-way ANOVA, followed by Dunnett's multiple comparisons test. *, **, and *** indicate p < 0.05, 0.01, and 0.001 of the live cells compared to the control (0 µg/mL).

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... death in the EL-4 cells treated with FBZ and CHOP was analyzed in two ways. First, we morphologically observed the nuclear appearance via Hoechst 33342/PI staining and classified cell death as follows: early apoptosis, in which the nuclei begin to condense (condensed and segmented blue-stained nuclei; Hoechst + /PI − ); late apoptosis, observed as condensed nuclei with damaged cell membranes (condensed and segmented pink-stained nuclei; Hoechst + /PI + ); and necrosis, observed as damaged cell membranes and large or concentrated nuclei (pyknosis) (undivided pink-or red-stained nuclei; Hoechst +/− /PI + ) ( Figure 2A). Cell viability was reduced by 9%, 33%, and 73% at FBZ concentrations of 0.025 µg/mL, 0.05 µg/mL, and 0.1 µg/mL, respectively, and it was reduced by 28% at 0.3 µg/mL of CHOP compared to the control. ...

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... death in the EL-4 cells treated with FBZ and CHOP was analyzed in two ways. First, we morphologically observed the nuclear appearance via Hoechst 33342/PI staining and classified cell death as follows: early apoptosis, in which the nuclei begin to condense (condensed and segmented blue-stained nuclei; Hoechst + /PI − ); late apoptosis, observed as condensed nuclei with damaged cell membranes (condensed and segmented pink-stained nuclei; Hoechst + /PI + ); and necrosis, observed as damaged cell membranes and large or concentrated nuclei (pyknosis) (undivided pink-or red-stained nuclei; Hoechst +/− /PI + ) ( Figure 2A). Cell viability was reduced by 9%, 33%, and 73% at FBZ concentrations of 0.025 µg/mL, 0.05 µg/mL, and 0.1 µg/mL, respectively, and it was reduced by 28% at 0.3 µg/mL of CHOP compared to the control. ...

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... cells begin to undergo apoptosis, the phosphatidyl serine (PS) inside the cell is exposed to the outside through membrane changes. The exposed PS was stained with annexin V, and the cells with damaged membranes were double-stained with PI to observe cell death separately within the same number of cells ( Figure 2B). When treated with 0.1 µg/mL of FBZ and 0.3 µg/mL of CHOP, the proportion of live cells (annexin V − /PI − ) decreased by 33% and 24%, respectively, compared to that of the control, and the proportion of apoptotic cells (annexin V + /PI +/− ) increased by 31% and 15%, respectively. ...