TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

Hooijdonk, C.A.E.M. van; Glade, C.P.; Erp, P.E.J. van

doi:10.1002/cyto.990170212

Cited by 89 publications

(63 citation statements)

References 16 publications

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“…The level of TO-PRO-3 incorporation into cellular DNA is indicative of significant membrane injury and has been widely used to distinguish dead from living cells (Van Hooijdonk et al, 1994). Our previous results showed that pyramidal neuron survival was variable after a 15 min.…”

Section: To-pro-3 Iodide Staining and Quantification Of Neuronal Viabmentioning

confidence: 98%

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Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

Zhang

Schools²,

Lei

et al. 2008

Experimental Neurology

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Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4′-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemiainduced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using wholecell patch clamp recording. 100 μM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In the parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 μM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access INTRODUTIONRecent studies have shown that resveratrol (RVT) can protect ischemia damage in a variety of organs, such as brain, kidney and heart (Ray, et al., 1999; Bastianetto et al., 2000;Chander and Chopra, 2006). RVT is one of the primary active polyphenol compounds identified in red wine (Jang et al., 1997; Bastianetto et al., 2000;Oak et al., 2005), which has attracted increasing research interest on how this compound could offer such a miracle protection. Mechanistically, RVT acts as a potent antioxidant and scavenger of hydroperoxyl free radicals (Pace-Asciak et al., 1996; Mokni et al., 2006;Das et al., 2006). These protective actions of RVT in cerebral ischemia have so far been identified from studies in vivo and in cultured n...

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Section: To-pro-3 Iodide Staining and Quantification Of Neuronal Viabmentioning

confidence: 98%

“…It labels DNA in cells only with compromised cell membranes (Van Hooijdonk et al, 1994). Exposure to OGD for 20 min.…”

Section: Ogd Induced Acute Neuronal Death Was Largely Attenuated By Rvtmentioning

confidence: 99%

Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

Zhang

Schools²,

Lei

et al. 2008

Experimental Neurology

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“…After the last centrifugation and supernatant discard, cells were transferred to microtubes in a final volume of 250 L of PBS or To-Pro-3 iodide (Molecular Probes, Eugene, OR). The latter was used for viability gating as needed (10,11). The microtubes were inserted into corresponding 12 ϫ 75 mm tubes in the loader rack of a FACScalibur flow cytometer (Becton, Dickinson [BD], San Jose, CA) for data acquisition.…”

Section: Cell Surface Stainingmentioning

confidence: 99%

DRAQ5‐based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties

Yuan

Douglas-Nikitin

Ahrens

et al. 2004

Cytometry Part B Clinical

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Background: Analysis of cell cycle kinetics offers important information regarding the behavior of normal and neoplastic cells. Most often, cell cycle determinations by flow cytometry (FCM) have been performed using whole-sample analysis with intercalating dyes like propidium iodide (PI). The cell cycle phase assessment in individual cell subsets in heterogeneous samples is best performed using combined antigen/ scatter and DNA analysis. DRAQ5, a novel DNA binding dye that excites at 488 nm and emits in the far red spectra, rapidly penetrates intact live cells while preserving their light scatter properties and expression of surface antigens. We evaluated the ability of this dye to measure cell cycle phases in a variety of clinical hematolymphoid samples.Methods: We first compared whole sample DRAQ5 and PI cell cycle analyses in 26 clinical hematolymphoid samples. Next, we analyzed cell subpopulations in 39 samples of nonpathologic bone marrow by performing simultaneous CD45/CD34 and DRAQ5 staining. We assessed cell cycle characteristics specific to each population identified by CD45/CD34/side light scatter: lymphocytes, monocytes, immature and mature granulocytes, nucleated erythroid cells, and early precursors.Results: Whole sample DNA cell cycle analyses by DRAQ5 and PI showed no significant differences in S-phase. DRAQ5, however, produced slightly larger coefficients of variation. DRAQ5-based DNA content analysis was easily performed on the distinct marrow cell subpopulations, since light scatter and antigen expression were completely preserved. Significant differences in S-phase were noted between subpopulations of cells exhibiting different degrees of maturation.Conclusions: Because of its simplicity of use, excitability with 488 nm lasers, and the ability to stain viable cells, DRAQ5 should prove most useful in the kinetic evaluation of normal and neoplastic hematolymphoid cell subsets identified by light scatter and antigenic expression.

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“…TO-PRO-3 seems to be best suited for multicolor analysis, and already has been utilized for the multi-parameter flow cytometric analysis [6,7,27]. But, it has one shortcoming: fluorescence instability under laser illumination [18,24].…”

Section: Prevention Of the Photobleaching Of Dna-specific Fluorocmentioning

confidence: 99%

Fluorescence Counter-Staining of Cell Nuclear DNA for Multi-Color Laser Confocal Microscopy.

Suzuki

Matsuzaki

Takata

1998

Acta Histochem. Cytochem

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TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

Cited by 89 publications

References 16 publications

Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

DRAQ5‐based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties

Fluorescence Counter-Staining of Cell Nuclear DNA for Multi-Color Laser Confocal Microscopy.

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