Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye

Leggate, Johanna; Allain, Ray; Isaac, Leah; Blais, Burton W.

doi:10.1007/s10529-006-9128-1

Cited by 60 publications

(50 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Different phases of biofilm (8,12, and 24 h), with or without azetazolamide (128 mg/liter; Sigma), a chitinase inhibitor (36), were formed at 37°C either in microtiter plates or in tissue culture flasks as described above. The quantity of eDNA within the ECM was measured using a microplate fluorescence assay (MFA) with a DNA-binding dye (SYBR green I) as previously described (37). Briefly, SYBR green I (Invitrogen, Paisley, United Kingdom) was added to eDNA extract in a black-well microtiter plate (Costar, Corning, NY) at a ratio of 1:1.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

et al. 2013

View full text Add to dashboard Cite

dAspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.

show abstract

Section: Methodsmentioning

confidence: 99%

“…The levels of eDNA were quantified using a fluorescence plate reader (Fluostar Optima; BMG Labtech, Aylesbury, United Kingdom) at excitation and emission wavelengths of 485 and 518 nm, respectively. The concentration of eDNA in the sample was quantified by using a DNA standard curve as previously described elsewhere (37).…”

Section: Methodsmentioning

confidence: 99%

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

et al. 2013

View full text Add to dashboard Cite

show abstract

“…After culture, islets were preincubated for 45 min in a bicarbonate-buffered Krebs solution containing G0.5 and then incubated in batches of five for 1 h at various glucose concentrations. At the end, the medium was collected for insulin measurement, and the islet insulin and DNA contents were measured on each batch of islets (23). Insulin secretion was normalized for variations in islet DNA content.…”

Section: Methodsmentioning

confidence: 99%

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration

Roma

Duprez

Jonas³

2015

American Journal of Physiology-Endocrinology and Metabolism

View full text Add to dashboard Cite

Roma LP, Duprez J, Jonas JC. Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration. Am J Physiol Endocrinol Metab 309: E632-E639, 2015. First published August 11, 2015; doi:10.1152/ajpendo.00154.2015.-In rat pancreatic islets, ␤-cell gene expression, survival, and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here, we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. Rat pancreatic islets were cultured 1-7 days at G5, G10, or G30 with or without 3 M of the GK activator Ro 28-0450 (Ro). After culture, ␤-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H autofluorescence, intracellular calcium concentration, and insulin secretion were tested in the absence or presence of Ro. Prolonged culture of rat islets at G5 or G30 instead of G10 triggered ␤-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected ␤-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to ␤-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10 but failed at restoring ␤-cell glucose responsiveness after culture at G5 or G30. We conclude that pharmacological GK activation prevents the alteration of ␤-cell survival and function by long-term culture at G5 but mimics glucotoxicity when added to G10. The complex effects of glucose on the ␤-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se. apoptosis; glucotoxicity; insulin secretion; pancreatic ␤-cell GLUCOSE STIMULATION OF INSULIN SECRETION (GSIS) by endocrine pancreatic ␤-cells depends on the acceleration of glucose metabolism through glycolysis and the TCA cycle, with enhanced production of metabolic coupling factors (25,39,41). Besides these rapid effects, glucose exerts complex long-term effects on the ␤-cell phenotype (2,10,16,18,27,45). During long-term culture of rodent islets, ␤-cell gene expression, survival, and glucose responsiveness are optimally preserved in the presence of 10 mM glucose (G10), whereas they are markedly altered by culture at either nonstimulating (G5) or very high (G30) glucose concentrations. In other words, culture at G10 vs. G5 is beneficial, whereas culture at G30 vs. G10 is detrimental to ␤-cell gene expression, survival, and glucose responsiveness. The beneficial effect of culture at G10 vs. G5 is usually attributed to the stimulation of energetic metabolism. In contrast, the deleterious effects of culture at G30 vs. G10, which we later refer to as glucotoxicity, could result from the further increase in metabolism with increased production o...

show abstract

“…Mono-and oligonucleosomes present in the cytosolic fraction were measured in duplicates. The mean absorbance of each sample was normalized to that of the positive control provided in the kit and corrected for differences in islet DNA content measured (in the nuclear fraction) by fluorimetry using SYBR Green I (25). The percentage of apoptotic islet cells was also determined using the in situ cell death detection kit (Roche Diagnostics).…”

Section: Methodsmentioning

confidence: 99%

“…The acute glucose-induced changes in intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) were recorded by microspectrofluorimetry in furaPE3-loaded islets while glucosestimulated insulin secretion was measured in 2-min effluent collections from 25-100 perifused islets by RIA, as described previously (34). After perifusion the islets were disrupted by sonication in TNE (10 mmol/l Tris, 0.2 mmol/l NaCl, 10 mmol/l EDTA), and their insulin and DNA contents were measured (25).…”

Section: Measurements Of Intracellular Camentioning

confidence: 99%

Effects of fructosamine-3-kinase deficiency on function and survival of mouse pancreatic islets after prolonged culture in high glucose or ribose concentrations

Pascal

Veiga‐da‐Cunha

Gilon³

et al. 2010

American Journal of Physiology-Endocrinology and Metabolism

View full text Add to dashboard Cite

Pascal SM, Veiga-da-Cunha M, Gilon P, Van Schaftingen E, Jonas JC. Effects of fructosamine-3-kinase deficiency on function and survival of mouse pancreatic islets after prolonged culture in high glucose or ribose concentrations. Am J Physiol Endocrinol Metab 298: E586 -E596, 2010. First published December 15, 2009 doi:10.1152/ajpendo.00503.2009.-Due to their high glucose permeability, insulin-secreting pancreatic ␤-cells likely undergo strong intracellular protein glycation at high glucose concentrations. They may, however, be partly protected from the glucotoxic alterations of their survival and function by fructosamine-3-kinase (FN3K), a ubiquitous enzyme that initiates deglycation of intracellular proteins. To test that hypothesis, we cultured pancreatic islets from Fn3k-knockout (Fn3k Ϫ/Ϫ ) mice and their wild-type (WT) littermates for 1-3 wk in the presence of 10 or 30 mmol/l glucose (G10 or G30, respectively) and measured protein glycation, apoptosis, preproinsulin gene expression, and Ca 2ϩ and insulin secretory responses to acute glucose stimulation. The more potent glycating agent D-ribose (25 mmol/l) was used as positive control for protein glycation. In WT islets, a 1-wk culture in G30 significantly increased the amount of soluble intracellular protein-bound fructose-ε-lysines and the glucose sensitivity of ␤-cells for changes in Ca 2ϩ and insulin secretion, whereas it decreased the islet insulin content. After 3 wk, culture in G30 also strongly decreased ␤-cell glucose responsiveness and preproinsulin mRNA levels, whereas it increased islet cell apoptosis. Although protein-bound fructose-ε-lysines were more abundant in Fn3k Ϫ/Ϫ vs. WT islets, islet cell survival and function and their glucotoxic alterations were almost identical in both types of islets, except for a lower level of apoptosis in Fn3k Ϫ/Ϫ islets cultured for 3 wk in G30. In comparison, D-ribose (1 wk) similarly decreased preproinsulin expression and ␤-cell glucose responsiveness in both types of islets, whereas it increased apoptosis to a larger extent in Fn3k Ϫ/Ϫ vs. WT islets. We conclude that, despite its ability to reduce the glycation of intracellular islet proteins, FN3K is neither required for the maintenance of ␤-cell survival and function under control conditions nor involved in protection against ␤-cell glucotoxicity. The latter, therefore, occurs independently from the associated increase in the level of intracellular fructose-ε-lysines.

show abstract

Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye

Cited by 60 publications

References 9 publications

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration

Effects of fructosamine-3-kinase deficiency on function and survival of mouse pancreatic islets after prolonged culture in high glucose or ribose concentrations

Contact Info

Product

Resources

About