Fig. 1: Changes in cell number are governed by alterations in proliferation and cell death.

Changes in cell number are governed by alterations in proliferation and cell death. Cell death is most commonly associated with apoptosis, which progresses through early and late stages, distinguished by the intact or permeable status of cell membranes, respectively. Apoptosis is often triggered by mitochondrial depolarization and the subsequent release of cytochrome c. Thus, mitochondrial depolarization is linked to both the induction of apoptosis and a reduction in ATP production, as mitochondrial energy production relies on the mitochondrial membrane potential. Conversely, cell proliferation drives increases in cell numbers. Proliferation is characterized by the rate at which cells progress through the stages of the cell cycle, culminating in mitosis. Cells often spend a considerable amount of time in the S phase, and the rate of DNA synthesis during this phase can provide insights into the overall cellular status and energy balance. The protocol described in this paper integrates multiple flow cytometry stainings, enabling the evaluation of all these parameters.