Dead Cert: Measuring Cell Death.

Many cells in the body die at specific times to facilitate healthy development or because they have become old, damaged, or infected. Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals.

Analysis of Cytochrome c Release by Immunocytochemistry.

Cytochrome c is normally localized between the inner and outer membranes of mitochondria in healthy cells. However, during apoptosis, it is released into the cytoplasm, where it binds to apoptotic protease activating factor. Caspase-9 is then recruited and activated by this complex in a process known as the induced proximity model.

Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry.

The surface of healthy cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflet of the plasma membrane. One of these lipids, phosphatidylserine (PS), is normally restricted to the inner leaflet of the plasma membrane and is, therefore, only exposed to the cell cytoplasm. However, during apoptosis lipid asymmetry is lost and PS becomes exposed on the outer leaflet of the plasma membrane.

Detection of DNA Fragmentation in Apoptotic Cells by TUNEL.

Degradation of DNA into oligonucleosomal-sized fragments is a unique event in apoptosis that is orchestrated by caspase-activated DNase. Traditionally, this event is observed by resolving cellular DNA by gel electrophoresis, which results in a characteristic "ladder" pattern. However, this technique is time-consuming and cannot be used to quantitate the number of apoptotic cells in a sample.

Analyzing Cell Death by Nuclear Staining with Hoechst 33342.

The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells.

Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining.

Identifying and characterizing different forms of cell death can be facilitated by staining internal cellular structures with dyes such as hematoxylin and eosin (H&E). These dyes stain the nucleus and cytoplasm, respectively, and optimized reagents (e.g.

Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry.

Propidium iodide (PI) is a small fluorescent molecule that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane. PI uptake versus exclusion can be used to discriminate dead cells, in which plasma membranes become permeable regardless of the mechanism of death, from live cells with intact membranes. PI is excited by wavelengths between 400 and 600 nm and emits light between 600 and 700 nm, and is therefore compatible with lasers and photodetectors commonly available in flow cytometers.

Measuring Cell Death by Trypan Blue Uptake and Light Microscopy.

Trypan blue is a colorimetric dye that stains dead cells with a blue color easily observed using light microscopy at low resolution. The staining procedure is rapid and cells can be analyzed within minutes. The number of live (unstained) and dead (blue) cells can be counted using a hemocytometer on a basic upright microscope.

Triggering Apoptosis in Hematopoietic Cells with Cytotoxic Drugs.

Cytotoxic agents are commonly added to cultured cells in the laboratory to investigate their efficacy, mechanism of action, and therapeutic potential. Most of these agents trigger cell death by apoptosis, which is also the most common form of cell death during development, aging, homeostasis, and eradication of disease. Treatment of cells with cytotoxic agents is therefore useful for investigating basic mechanisms of cell death in the human body.

Global gene expression profiling of monocyte-derived macrophages from red deer (Cervus elaphus) genotypically resistant or susceptible to Mycobacterium avium subspecies paratuberculosis infection.

Mycobacterium avium subspecies paratuberculosis (MAP) can cause a chronic inflammatory bowel disease, Johne's disease (JD), in ruminant animals. This study has explored the molecular basis of resistance and susceptibility to this disease in red deer breeds previously confirmed to express polarised phenotypes by experimental infection trials and following natural infection. Monocyte-derived macrophage cultures were obtained from uninfected red deer selected for either a resistant or susceptible phenotype.