1 h 2 h 3 h 4 h

Figure 9-8 Percentage gated and mean cell volume (MCV) for FasL- and UV-treated DiBAC4-stained cells over time. Under each apoptotic condition, an increase in the number of DiBAC4-positive cells, along with a simultaneous decrease in the number of normal (DiBAC4 negative) cells, was observed over a period of 4 h. In contrast, no change in MCV was observed for either FasL- or UV-treated normal or DiBAC4-positive populations of cells, suggesting that the change in MCV remains constant over this period of time.

response to either FasL or UV treatment. Here, gates were set on an EV versus DiBAC4 fluorescence dot plot to analyze both the percentage of cells and the change in MCV for each population of cells. A constant percentage of normal control cells is observed over the 4-h time span (Fig. 9.7).

Additionally, there is no change in the MCV for the small percentage of DiBAC4-positive cells observed in the control sample. Upon either FasL or UV treatment, an increasing number of cells are observed in a time-dependent manner for the DiBAC4-positive cells (Fig. 9.8). However, the MCV remains constant regardless of the total number of cells examined. Thus, the Cell Lab Quanta SC can also be used to analyze populations of cells over time in regards to AVD during apoptosis.

11. Conclusion

The loss of cell volume during apoptosis is a fundamental characteristic of this cell death process. We have shown how AVD can be determined for various subpopulations of apoptotic cells using the Cell Lab Quanta SC flow cytometer. This instrument electronically sizes cells in conjunction with numerous fluorescent parameters to determine precise changes in cell diameter and cell volume. The application of these studies is not limited to only the examination of the externalization ofphosphatidylserine, caspase activity, and plasma membrane depolarization, but can be applied to other apoptotic characteristics that can be analyzed through the use of fluorescence.

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