Analyzing Cell Death Events in Cultured Leukocytes

Cell death is of utmost importance in immunity, in part as a way to control the development and activity of leukocytes, but also as a strategy employed by leukocytes to rid the body of unwanted cells. Apoptosis is the classic type of programmed cell death involving an ordered sequence of cellular events, resulting in morphological changes that include cleavage/fragmentation of DNA, condensation of nuclei, cell shrinkage, and alterations of the plasma membrane. The apoptotic cell is a nonfunctional, but structurally intact, entity with preserved membrane integrity that is engulfed by surrounding cells (a process known as clearance) in an immunologically silent manner. In contrast, necrotic cells, i.e., nonfunctional cells that have lost membrane integrity, are freely permeable and leak intracellular constituents that may shift immunological homeostasis. Thus, membrane integrity of dead leukocytes is very important from an immunological point of view. For the analysis of leukocyte cell death, a wide variety of assays are available to monitor different events along the cell death pathway; a combination of different methods is advantageous in order to gain a more complete understanding of this dynamic process. In this chapter, we describe several in vitro methods for evaluating leukocyte cell death, mainly focusing on apoptosis in human neutrophils and lymphocytes. Special emphasis is given to assessment of membrane integrity of the cultured cells. Furthermore, a protocol for monitoring clearance of apoptotic neutrophils by monocyte-derived macrophages is provided.