Identification of Neural Programmed Cell Death Through the Detection of DNA Fragmentation In Situ and by PCR

Abstract

A universal feature in the development of multicellular organisms is a physiological form of cell death called programmed cell death (PCD). A subset of PCD is apoptosis, which is defined by characteristic morphological changes and genomic DNA fragmentation producing what are referred to as nucleosomal ladders. To understand how PCD operates in a developing tissue or in a tissue following an experimental procedure, dying cells must be identified in relation to their surviving neighbors. One way to accomplish this is to visualize fragmented DNA in situ, in conjunction with gel electrophoresis of isolated DNA to visualize the nucleosomal ladders associated with apoptosis. Two approaches are presented in this unit: in situ end-labeling plus (ISEL+), a technique to identify dying cells in tissue sections or cell cultures of central nervous system (CNS) tissue (optimized for embryonic samples); and the use of ligation-mediated polymerase chain reaction (LMPCR) to identify nucleosomal ladders from intact tissues. Also included are procedures for preparing thymocyte cell cultures for use as controls in the ISEL+ procedure and for isolating genomic DNA for LMPCR.