Cytometry in cell necrobiology revisited. Recent advances and new vistas

Wlodkowic, Donald; Skommer, Joanna; Darzynkiewicz, Zbigniew

doi:10.1002/cyto.a.20889

Cited by 79 publications

(125 citation statements)

References 134 publications

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“…The cell can responds to the chemicals and microparticles by undergoing apoptosis, necrosis, or other cellular changes (53). There is a need to understand these cellular processes that are stimulated by nanoparticles, microparticles, and chemicals.…”

Section: Discussionmentioning

confidence: 99%

Detection of TiO₂ nanoparticles in cells by flow cytometry

et al. 2010

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Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO 2 nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human-derived retinal pigment epithelial cells (ARPE-19) were incubated with TiO 2 nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 lg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur TM flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO 2 . From 0.1 to 30 lg/ml TiO 2 , the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO 2 particles. Based on the parameters of morphology and the calcein-AM/propidium iodide viability assay, TiO 2 concentrations below 30 lg/ml TiO 2 caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E-800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO 2 (0.1-0.3 lg/ml), the flow cytometer could detect as few as 5-10 nanoparticles per cell due to intense light scattering by TiO 2 . Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health. Published

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Section: Discussionmentioning

confidence: 99%

Detection of TiO₂ nanoparticles in cells by flow cytometry

et al. 2010

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“…Imaging of various phenomena involving intracellular vesicles requires various image recording regimes-some of these processes are relatively fast, like vesicle fusion or "kiss-andrun" (5), while some are relatively slow, for instance digestion of cellular components in a process of autophagy (6,7). In the first case, a high number of image frames need to be collected over a short period of time, while in the second, although the frequency of image frames may be significantly lower, the overall time of the imaging needs to be long and extend into hours.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long‐term tracking of acidic vesicles

2014

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Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis. V C 2014International Society for Advancement of Cytometry

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“…Mechanical cell degradation and lysis of mitotic cells or micronuclei may appear as apoptotic cells. In addition to DNA deficit, other parameter should be used to positively identify apoptotic cells (9). We conclude that measuring the fraction of cells with sub-G 1 DNA content is useful for determining whether certain treatments induce apoptosis but is not an accurate quantitative measure of apoptosis and can be misleading regarding the relationship between apoptosis and cell cycle progression.…”

Section: Resultsmentioning

confidence: 98%