Detection of Apoptosis-like Cell Death in Ustilago maydis by Annexin V-FITC Staining.

Programmed cell death (PCD) guides the transition between key developmental stages in many organisms. PCD also remains an important fate for many organisms upon exposure to different stress conditions. Therefore, an insight into the progression of PCD during the execution of a biological phenomenon can yield significant details of the underlying mechanism. Apoptosis, as well as apoptosis-like programmed cell death, constitutes one of the forms of PCD in higher and lower eukaryotes respectively. Flipping of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer leaflet is among the different hallmarks of apoptosis/apoptosis-like PCD that marks the initiation of the said cell death event. This flipping can be detected through staining of the target cells using annexin V-FITC that binds specifically to PS. In Ustilago maydis the staining of the externally exposed PS by annexin V-FITC is difficult due to the presence of cell wall. The key to such staining, therefore, relies on the gentle removal of the cell wall without significantly altering the underlying plasma membrane architecture/topology. This protocol highlights the dependence of the PS staining on the extent of protoplastation of the stressed cells in Ustilago maydis.