The Pros and Cons of Apoptosis Assays for Use in the Study 
of Cells, Tissues, and Organs

Watanabe, Michiko; Hitomi, Midori; Wee, Kathy Van der; Rothenberg, Florence; Fisher, Steven A.; Zucker, Robert M.; Svoboda, Kathy K.H.; Goldsmith, Edie C.; Heiskanen, Kaisa M.; Nieminen, Anna‐Liisa

doi:10.1017/s1431927602010346

Cited by 121 publications

(110 citation statements)

References 36 publications

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“…1). LTR accumulates within dead and dying cells, allowing us to identify areas of cell death in whole-mount embryos, and has been used previously to study apoptosis in the embryonic mouse (Zucker et al, 1998;Watanabe et al, 2002).…”

Section: Neural Crest Cells Arementioning

confidence: 99%

Temporal–spatial ablation of neural crest in the mouse results in cardiovascular defects

Porras

Brown

2007

Developmental Dynamics

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Neural crest cells are thought to play a critical role in human conotruncal morphogenesis and dysmorphogenesis. Much of our understanding of the contribution of neural crest to cardiovascular patterning comes from ablation and transplantation experiments in avian species. Although fate mapping experiments in mice suggests a conservation of function, the functional requirement for neural crest in cardiovascular development in mammals has not been formally tested. We used a novel two component genetic system for the temporal-spatial ablation of neural crest in the mouse. Affected embryos displayed a spectrum of cardiovascular outflow tract defects and aortic arch patterning abnormalities. We show that the severity of the cardiovascular phenotype is directly related to the level and extent of neural crest ablation. This is the first report of cardiac neural crest ablation in mammals, and it provides important insight into the role of the mammalian neural crest during cardiovascular development. Developmental Dynamics 237:153-162, 2008.

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Section: Neural Crest Cells Arementioning

confidence: 99%

Temporal–spatial ablation of neural crest in the mouse results in cardiovascular defects

Porras

Brown

2007

Developmental Dynamics

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“…5, 2006 THYMUS HISTOPATHOLOGY 661 FIGURE 4.-(Continued) in the number of apoptotic cells should be noted only after comparison with controls. Although various methodologies are available for evaluating apoptosis (stained resin sections, DNA laddering, TUNEL, annexin V, caspase-3 activity assays, mitochondrial assays, vital dyes and lysotracker red), each assay has its advantages and disadvantages that can render it appropriate and useful for one application but inappropriate or difficult to use in another (Watanabe et al, 2002). Transmission electron microscopy ( Figure 2) is considered the "gold standard" for the evaluation of apoptosis.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Histopathology of the Thymus

Elmore

2006

Toxicol Pathol

112

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The thymus is a primary or central lymphoid organ in which T lymphocytes undergo diffentiation and maturation autonomously within the cortex, without the need for antigenic stimulation, and it is essential for the normal development and function of the immune system. The thymus has been shown to be a sensitive target organ following exposure to immunotoxicants and endogenous corticosteroids, and a decrease in size or weight is often one of the first noted measures of compound-induced effects with cortical lymphocytes (thymocytes) being especially susceptible. Therefore, changes in thymus histopathology and architecture are considered to be of particular relevance for immunotoxicity screening. The separate compartments in each lymphoid organ should be evaluated separately and descriptive rather than interpretive terminology should be used to characterize changes within those compartments (Haley et al., 2005). Therefore, enhanced histopathological evaluation of the thymus involves the determination of the size and cellularity of the cortex and medulla, which should be noted separately. Other changes to evaluate include, but are not limited to, increased lymphocyte apoptosis, lymphocyte necrosis, cortex:medulla ratio and an increase or decrease in the epithelial component of the thymus.

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“…1) This staining pattern correlates to the spatial location of cell death staining with classical vital dyes (e.g. Nile blue, neutral red, acridine orange) and other apoptotic detection methods used to determine cell death during normal embryological development or embryological toxicology (22,23,(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Sulik and coworkers have compared NBS and LT stains and shown that they stain the same regions (e.g.…”

Section: Lt Correlation With Cell Deathmentioning

confidence: 95%

Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis

Zucker

2006

Cytometry Pt A

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Background:After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three‐dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays.Methods:The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB.Results:Tissues as thick as 500 μm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide).Conclusions:The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red‐emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were ∼500‐μm thick. © 2006 International Society for Analytical Cytology

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The Pros and Cons of Apoptosis Assays for Use in the Study of Cells, Tissues, and Organs

Cited by 121 publications

References 36 publications

Temporal–spatial ablation of neural crest in the mouse results in cardiovascular defects

Temporal–spatial ablation of neural crest in the mouse results in cardiovascular defects

Enhanced Histopathology of the Thymus

Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis

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