by FACS (Fig. A.36). Annexin V staining is often combined with a viability dye such as propidium iodide (PI) or 7-aminoactinomycin D (7-AAD). These two dyes fluoresce when bound to DNA, but are unable to enter viable or apoptotic cells prior to their loss of membrane integrity. Therefore, when combined with Annexin V, cells in the early stages of apoptosis can be identified as Annexin V-positive but PI/7-AAD-negative, whereas cells in the late stages of apoptosis are both Annexin V-positive and PI/7-AAD-positive. An additional assay that provides a sensitive means of detecting apoptotic cells by FACS analysis is based on the detection of activated caspase 3, a cysteine protease that functions in the execution phase of the apoptotic cell death program. Caspase 3 is initially synthesized by cells in an inactive precursor form called a pro-caspase. When cells are undergoing apoptosis, pro-caspase 3 is cleaved into two subunits that dimerize to form the active enzyme. Antibodies have been generated that detect the active form of caspase 3, but not pro-caspase 3, and fluorescently coupled versions of these antibodies can be used to detect apoptotic cells that have been fixed and permeabilized (Fig A.37). A-28 Assays for cytotoxic T cells. Activated CD8 T cells generally kill any cells that display the specific pep-tide:MHC class I complex they recognize. Thus, CD8 T-cell function can be determined using the simplest and most rapid T-cell bioassay—the killing of a target cell by a cytotoxic T cell. This is usually detected in a 51Cr-release assay. Live cells will take up, but do not spontaneously release, radioactively labeled sodium chromate, Na2 51CrO4. When these labeled cells are killed, the radio-active chromate is released and its presence in the supernatant of mixtures of target cells and cytotoxic T cells can be measured (Fig. A.38). In a similar assay, proliferating target cells such as tumor cells can be labeled with 3H-thymidine, which is incorporated into the replicating DNA. On attack by a cytotoxic T cell, pro-caspase 3/7 active caspase 3/7 pro-caspase 9 caspase 9 cytochrome c Apoptosis Apaf-1 active Apaf-1 Apoptotic stimuli activate caspase 3/7 Specifc antibody binds only to active caspase 3/7 Fig. A.37 Detection of apoptotic cells by intracellular staining for active caspases. An early event in the apoptotic process is the release of cytochrome c from mitochondria. Cytochrome c binds to Apaf-1, leading to the cleavage of pro-caspase 9 into active caspase 9. Caspase 9 then cleaves pro-caspase 3 and pro-caspase 7 to yield their active forms, ‘executioner’ caspases that promote cell death. Antibodies that recognize the active caspase 3 or caspase 7, but not the pro-caspase forms of these enzymes, will detect permeabilized cells undergoing apoptosis. Fig. A.36 Detection of apoptotic cells with Annexin V. In healthy cells, the membrane phospholipid phosphatidylserine is oriented with its polar headgroup facing the cytosolic face of the plasma membrane. When cells undergo apoptosis, the enzyme responsible for maintaining phosphatidylserine polarity, called flippase, is no longer active. As a result, phosphatidylserine becomes randomly oriented, with many molecules exposing their polar head groups on the extracellular face of the plasma membrane. The protein Annexin V binds tightly to the exposed phosphatidylserine and, if fluorescently labeled, can be used to detect apoptotic cells by FACS. healthy cell In an apoptotic cell phosphatidylserine appears on the outer leafet of the plasma membrane and will bind to Annexin V  In a healthy cell phosphatidylserine is confned to the inner leafet of the plasma membrane phosphatidyl-ethanolamine Annexin V phosphatidyl serine phosphatidyl choline apoptotic cell