Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cellsin vitro

Räthel, Thomas; Leikert, Jürgen F.; Vollmar, Angelika M.; Dirsch, Verena M.

doi:10.1251/bpo55

Cited by 74 publications

(65 citation statements)

References 36 publications

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“…The finding that this background emission is equivalent to the activation of 2 µM of DAF-2-T within the tissue attests to its significance and potential for interfering with the detection of DAF-2 T formation. The importance of DAF-2 autofluorescence was first pointed out by Leikert et al [9] and Rathel et al [10] in the context of DAF-2 in tissue perfusates, which our results now extend to tissue NO bioimaging.…”

Section: What Is the True Sensitivity Of Daf-2 For Imaging No Sourcessupporting

confidence: 79%

“…A number of studies have reported on the nitrosation of DAF-2 for varying concentrations of NO donors, either in buffer solutions [9,10,20,21,30] using fluorometry or in cells/tissues [26,29] using fluorescence microscopy. …”

Section: Comparative Nitrosation Of Daf-2 In Buffer Solutions and Tismentioning

confidence: 99%

“…This chemical modification of the dye by a secondary product of NO is associated with a lowering of the energetic charge transfer state below the ground state of the fluorophore, thus removing the quenching effect of the benzoic structure and allowing the fluorophore to emit efficiently. The high yield of fluorescence of the triazole forms of DAF and DAR make these indicators suitable for the detection of NO production in biological systems, a property that has been exploited by now in many studies using fluorometry [6][7][8][9][10][11][12][13][14][15][16][17], flow cytometry [18,19], high-pressure liquid chromatography (HPLC) [20], and fluorescence microscopy [4,14,. Furthermore, when used in conjunction with other probes, diamino fluorophores theoretically provide the option to test for co-localization of NO formation with other signaling events, such as a change in intracellular calcium concentration using Fura-2 [12,16,38], or its association with certain cell organelles by e.g.…”

Section: Introductionmentioning

confidence: 99%

“…These new findings add to the growing body of knowledge on how to best optimize their use [9,10] and avoid potential pitfalls [62][63][64][65][66], which will ultimately help investigators to use this relatively new class of fluorophores more judiciously for the investigation of NO-related events.…”

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confidence: 99%

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Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Rodríguez

Specian

Maloney

et al. 2005

Free Radical Biology and Medicine

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An emergent approach to the detection of nitric oxide (NO) in tissues relies on the use of fluorescence probes that are activated by products of NO autoxidation. Here we explore the performance of the widely-used NO probe 4,5-diaminofluorescein diacetate (DAF-2 DA) for the localization of sources of NO in rat aortic tissue, either from endogenous NO synthesis or from chemically or photolytically-released NO from targets of nitrosation/nitrosylation. Of importance toward understanding the performance of this probe in tissues is the finding that, with incubation conditions commonly used in the literature (10 µM DAF-2 DA), intracellular DAF-2 accumulates to concentrations that approach the millimolar range. Whereas such high probe concentrations do not interfere with NO release or signaling, they help to clarify why DAF-2 nitrosation is possible in the presence of endogenous nitrosation scavengers (e.g. ascorbate and glutathione). The gain attained with such elevated concentrations is, however, mitigated by associated high levels of background autofluorescence from the probe. This, together with tissue autofluorescence, limits the sensitivity of the probe to low-micromolar levels of accumulated DAF-2 triazole (DAF-2 T), the activated form of the probe, which is higher than the concentrations of endogenous nitrosation/nitrosylation products found in tissues. We further show that the compartmentalization of DAF-2 around elastic fibers further limits its potential to characterize the site of NO production at the subcellular level. Moreover, we find that reaction of DAF-2 with HgCl 2 and other commonly employed reagents are associated with spectral changes that may be misinterpreted as NO signals. Finally, UV illumination can lead to high levels of nitrosating species that interfere with NO detection from enzymatic sources. These findings indicate that while DAF-2 may still represent an important tool for the localization of NO synthesis, provided important pitfalls and limitations are taken into consideration, it is not suited for the detection of basally-generated nitrosation/nitrosylation products.Performance of DAF-2 for NO Imaging 3

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Section: What Is the True Sensitivity Of Daf-2 For Imaging No Sourcessupporting

confidence: 79%

Section: Comparative Nitrosation Of Daf-2 In Buffer Solutions and Tismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Rodríguez

Specian

Maloney

et al. 2005

Free Radical Biology and Medicine

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“…Of these analyses however, only fluorometric and electrochemical have both the sensitivity and flexibility to measure low levels of constitutively produced NO from cultured cells in situ. Particularly, the fluorescent indicator diaminofluorescein (DAF) has been used to quantify NO generated by cultured myofibroblasts [21] [22]. While this method is highly sensitive, it has had limited success due to the susceptibility of the calibration standard to photolysis.…”

Section: Introductionmentioning

confidence: 99%

An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts

Sharma¹,

Rowland²,

Clouse³

et al. 2014

ABC

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Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NO X ) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.

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A Nanocoating Co‐Localizing Nitric Oxide and Growth Factor onto Individual Endothelial Cells Reveals Synergistic Effects on Angiogenesis

Jeong

Choi

et al. 2021

Adv Healthcare Materials

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The delivery of nitric oxide (NO)-an intrinsic cellular signaling molecule-is promising for disease treatment, in particular to vascular diseases, due to its endothelial-derived inherent nature. The limited diffusion distance of labile NO prompts researchers to develop various carriers and targeting methods for specific sites. In contrast to the apoptotic effect of NO, such as anticancer, delivering low NO concentration at the desired targeting area is still intricate in a physiological environment. In this study, the layer-by-layer assembled nanocoating is leveraged to develop a direct NO delivery platform to individual endothelial cells (ECs). NO can be localized to individual ECs via S-nitrosothiol-bound polyacrylic acid which is a polymer directly providing an endothelial-like constant level of NO. To increase angiogenic activation along with NO, VEGF is additionally applied to specific receptors on the cell surface. Notably, the survival and proliferation of ECs are significantly increased by a synergistic effect of NO and VEGF co-localized via nanocoating. Furthermore, the nanocoating remarkably promoted cell migration and tubule formation-prerequisites of angiogenesis. The proposed unique technology based on nanocoating demonstrates great potential for conferring desired angiogenic functions to individual ECs through efficient NO delivery.

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Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cellsin vitro

Cited by 74 publications

References 36 publications

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts

A Nanocoating Co‐Localizing Nitric Oxide and Growth Factor onto Individual Endothelial Cells Reveals Synergistic Effects on Angiogenesis

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