Aging Aging

72 Carmody et al.This chapter outlines some current procedures for the detection of apoptosisand the analysis of intracellular molecular events important in apoptotic pathways.Biochemical events include the generation of reactive oxygen species(ROS) and disruption of mitochondrial transmembrane potential (∆ψ m ). Themethods described in this chapter all utilize a flow cytometer for quantitativeanalysis of data. Several techniques (e.g., propidium iodide [PI] or terminaldeoxyuridine triphosphate [dUTP] nick end labeling [TUNEL]) assay may, however,be adapted for use with a fluorescent microscope. Flow cytometric analysishas the advantage of a rapid assessment of large numbers of cells in a highlyquantitative, nonsubjective manner. In addition, flow cytometry enables the parallelassessment of two and possibly three parameters in the same cell. Cellsorting may also be an option for some users (see also subsequent sections).1.1. Translocation of PhosphatidylserineSeveral studies have shown that phosphatidylserine (PS) is asymmetricallydistributed and is preferentially located in the inner leaflet of the plasma membrane(PM) (5). In the early stages of apoptosis in virtually all cell types, redistributionof membrane phospholipids results in the exposure of PS on the outermembrane (6). PS exposure has been shown to be tightly coupled to otherapoptosis-associated changes (7), and seems to be stimulus independent (6).Externalization of PS, following the induction of apoptosis, can be readilydetected using recombinant Annexin V, a protein that binds with high affinityto PS in a Ca 2+ -dependent manner (8). Because necrotic cells have lost theirmembrane integrity, they may also stain positive with Annexin V. However,dual staining with PI enables membrane-disrupted cells to be readily distinguished.Secondary necrotic cells are apoptotic cells which have subsequentlylost membrane integrity (see Subheading 4.1.1.), and therefore cannot bedistinguised from primary necrotic cells using this method.As Annexin V detects a cell surface marker, no fixation or permeabilizationis required for the procedure. The cells are therefore analyzed “alive” and maybe recovered by fluorescence-activated cell sorter (FACS) sorting for furtheranalytical purposes. This method also facilitates dual labeling for surface antigensthat recognize native antigen conformations.1.2. DNA FragmentationThe degradation of DNA into oligonucleosomal sized fragments of 180–200basepairs by specific endonuclease activity is a major biochemical event duringapoptosis in most cell types (9). This DNA fragmentation was originallyobserved as a ladder pattern using agarose gel electrophoresis. However, thismethod of detection is essentially qualitative and does not allow for the identificationof subpopulations of apoptotic cells. Several flow cytometric methods