Continuous Exposure to Low Doses of Ultrafine Black Carbon Reduces the Vitality of Immortalized Lung-Derived Cells and Activates Senescence

Figure 3

UBC induces low to moderate apoptotic conditions, as shown by FITC-Annexin V/PI costaining. All cells were seeded on day zero then treated with three concentrations of UBC, as described in Methods. Following day 9, the cells were harvested and apoptosis/necrosis was assessed by flow cytometry after double staining with FITC-Annexin V and PI. Percentage of apoptotic cells are shown for each of the cell lines ((a), (b), (c), and (d)) upon treatment with the highest UBC concentration. A representative double-parameter flow cytometry dot-plot for RAW 264.7 is presented (e) where N1 shows necrotic cells that are PI-positive, but FITC-Annexin V negative; N2 depicts late apoptotic cells that are FITC-Annexin V and PI double-positive; N3 specifies unstained viable cells with intact membranes that are FITC-Annexin V and PI double-negative; N4 contains early apoptotic cells that are PI-negative yet FITC-Annexin V positive. Dots (or events) show density quadrants. Low-density regions are designated in a lighter number of events, high-density regions in the darker number of events. Approximately 10,000 events were acquired per sample. Data determined as the mean of six replicates and is representative of three independent experiments. Statistically significant at in comparison to media (untreated) controls.

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