Measuring cell viability with membrane impermeable zinc fluorescent indicator

Stork, Christian J.; Li, Yang V.

doi:10.1016/j.jneumeth.2005.12.029

Cited by 23 publications

(19 citation statements)

References 22 publications

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“…We found a strong correlation between FJ and the Zn 2+ indicator dyes (NG, FZ3, RZ3, and TSQ). In this respect, our findings support the work of Stork and Li (2006) who reported the co-localization of the cell viability stain, propidium iodide, with a Zn 2+ indicator dye, NG (Stork and Li, 2006). We also found high correlation between FJ and the Ca 2+ indicator dye, Rhod-5N.…”

Section: Discussionsupporting

confidence: 91%

Fluorophilia: Fluorophore-containing compounds adhere non-specifically to injured neurons

et al. 2012

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Ionic (free) zinc (Zn2+) is implicated in apoptotic neuronal degeneration and death. In our attempt to examine the effects of Zn2+ in neurodegeneration following brain injury, we serendipitously discovered that injured neurons bind fluorescein moieties, either alone or as part of an indicator dye, in histologic sections. This phenomenon, that we have termed “fluorophilia”, is analogous to the ability of degenerating neuronal somata and axons to bind silver ions (argyrophilia — the basis of silver degeneration stains). To provide evidence that fluorophilia occurs in sections of brain tissue, we used a wide variety of indicators such as Fluoro-Jade (FJ), a slightly modified fluorescein sold as a marker for degenerating neurons; Newport Green, a fluorescein-containing Zn2+ probe; Rhod-5N, a rhodamine-containing Ca2+ probe; and plain fluorescein. All yielded remarkably similar staining of degenerating neurons in the traumatic brain-injured tissue with the absence of staining in our sham-injured brains. Staining of presumptive injured neurons by these agents was not modified when Zn2+ in the brain section was removed by prior chelation with EDTA or TPEN, whereas staining by a non-fluorescein containing Zn2+ probe, N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ), was suppressed by prior chelation. Thus, certain fluorophore-containing compounds nonspecifically stain degenerating neuronal tissue in histologic sections and may not reflect the presence of Zn2+. This may be of concern to researchers using indicator dyes to detect metals in brain tissue sections. Further experiments may be advised to clarify whether Zn2+-binding dyes bind more specifically in intact neurons in culture or organotypic slices.

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Section: Discussionsupporting

confidence: 91%

Fluorophilia: Fluorophore-containing compounds adhere non-specifically to injured neurons

et al. 2012

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“…The viability of cells under fluid shear stress conditions was determined by a combination of two fluorescent dyes, following established protocols (46): calcein acetoxymethyl (AM) (5 M) and propidium iodide (PI) (5 M) (AAT Bioquest). Calcein AM (green in color) and PI (red in color) were used to detect live and dead cells, respectively.…”

Section: Methodsmentioning

confidence: 99%

Microfluidic shear stress-regulated surfactant secretion in alveolar epithelial type II cells in vitro

Mahto

Tenenbaum-Katan

Greenblum

et al. 2014

American Journal of Physiology-Lung Cellular and Molecular Phys

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We investigated the role of flow-induced shear stress on the mechanisms regulating surfactant secretion in type II alveolar epithelial cells (ATII) using microfluidic models. Following flow stimulation spanning a range of wall shear stress (WSS) magnitudes, monolayers of ATII (MLE-12 and A549) cells were examined for surfactant secretion by evaluating essential steps of the process, including relative changes in the number of fusion events of lamellar bodies (LBs) with the plasma membrane (PM) and intracellular redistribution of LBs. F-actin cytoskeleton and calcium levels were analyzed in A549 cells subjected to WSS spanning 4-20 dyn/cm(2). Results reveal an enhancement in LB fusion events with the PM in MLE-12 cells upon flow stimulation, whereas A549 cells exhibit no foreseeable changes in the monitored number of fusion events for WSS levels ranging up to a threshold of ∼8 dyn/cm(2); above this threshold, we witness instead a decrease in LB fusion events in A549 cells. However, patterns of LB redistribution suggest that WSS can potentially serve as a stimulus for A549 cells to trigger the intracellular transport of LBs toward the cell periphery. This observation is accompanied by a fragmentation of F-actin, indicating that disorganization of the F-actin cytoskeleton might act as a limiting factor for LB fusion events. Moreover, we note a rise in cytosolic calcium ([Ca(2+)]c) levels following stimulation of A549 cells with WSS magnitudes ranging near or above the experimental threshold. Overall, WSS stimulation can influence key components of molecular machinery for regulated surfactant secretion in ATII cells in vitro.

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“…In slices that underwent OGD/R treatment, increased [Zn 2 + ] i was consistently observed in CA1 pyramidal neurons as shown by significantly increased NG fluorescence ( Figure 1B), supporting a robust increase in cytosolic labile Zn 2 + after injury. We also used other fluorescent Zn 2 + indicators (Zinpyr-1 and FluoZin-3) to confirm the increase in [Zn 2 + ] i (data not shown; but see Stork and Li, 2006b). Co-staining with the fluorescent cell death marker PI revealed that increased NG labeling occurred specifically in PI-positive, ischemically injured/dead pyramidal neurons ( Figure 1A and 1B).…”

Section: Ogd/r Neuronal Injury Is Coupled With Rising Zn 2 + or Zn 2 mentioning

confidence: 99%

Rising Zinc: A Significant Cause of Ischemic Neuronal Death in the CA1 Region of Rat Hippocampus

Stork

2009

J Cereb Blood Flow Metab

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There is a rising intracellular Zn 2 + transient during neuronal ischemic hypoxia (oxygen-glucose deprivation and reoxygenation, OGD/R). The results of our recent works suggest that the OGD/ R-induced Zn 2 + transient can readily be mistaken for a Ca 2 + transient. The aim of this study was to examine the respective functions of Zn 2 + and Ca 2 + in OGD/R-induced neuronal injury. We showed that [Zn 2 + ] i accumulation was consistently met with the induction of OGD/R-induced cell injury. Ca 2 + accumulation induced with high [K + ] (to open voltage-gated calcium channels) or ionomycin (a Ca 2 + ionophore) caused a moderate neuronal injury that was reduced significantly by the application of the Zn 2 + chelator N,N,N 0 ,N 0 -tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In comparison, Zn 2 + accumulation, induced with the Zn 2 + ionophore pyrithione, resulted in significantly greater injury. The application of nimodipine and MK801 was shown to attenuate neuronal injury only from a mild (10 mins) OGD insult. Neuronal injury from more severe (30 mins) OGD was not mitigated by the ion channel antagonists, whereas treatment with the Zn 2 + chelator TPEN did afford significant protection from cell injury. These results indicate Zn 2 + -mediated damage to be of greater consequence than Ca 2 + -mediated damage, and collectively support the suggestion that Zn 2 + accumulation may be a more significant causal factor of OGD/R-induced neuronal injury.

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Measuring cell viability with membrane impermeable zinc fluorescent indicator

Cited by 23 publications

References 22 publications

Fluorophilia: Fluorophore-containing compounds adhere non-specifically to injured neurons

Fluorophilia: Fluorophore-containing compounds adhere non-specifically to injured neurons

Microfluidic shear stress-regulated surfactant secretion in alveolar epithelial type II cells in vitro

Rising Zinc: A Significant Cause of Ischemic Neuronal Death in the CA1 Region of Rat Hippocampus

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