Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

Summary Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst-33342, 4’, 6-diamidino-2-phenylindole (DAPI), Acridine orange–Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin-V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells. Lay description The purpose of staining is to enable us to visualize the cellular and nuclear morphology during development and diseases. DNA is involved in the proper functioning of the cell and regulates numerous events. The changes in the integrity of DNA may lead to various disorders and diseases. The loss of functioning starts from the nuclear level and reaches up to the cellular level. Apoptosis, a programmed cell death is an important biological phenomenon and occurs in multicellular organisms including animals, plants and humans. Defective apoptotic processes are implicated in an extensive variety of diseases such as cancer, neurodegenerative, haematological, autoimmune and inflammatory diseases. Apoptosis is characterized by nuclear fragmentation, chromatin condensation and DNA laddering; therefore, analysis of nuclear events is an important step to understand the mechanism of diseases and development as well as their prevention. In view of such a critical role played by DNA, various microscopic techniques have been evolved over the year and used as potent diagnostic tools to identify the morphological and nuclear changes in cells. However, it is essential to understand the principle, application and precautions of various stains as they may have toxicity. This review is aimed to aptly summarize some of the most commonly used fluorescent and nonfluorescent microscopic techniques that shall assist researchers to understand and evaluate various techniques and use them for research.