scholarly journals Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 674
Author(s):  
Megan J. Reiniers ◽  
Lianne R. de Haan ◽  
Laurens F. Reeskamp ◽  
Mans Broekgaarden ◽  
Rowan F. van Golen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Data Interpretation ◽  
Medium Composition ◽  
Cultured Hepatocytes ◽  
Experimental Conditions ◽  
Membrane Transport Proteins ◽  
Dichlorofluorescein Diacetate ◽  
Cell Type Specific ◽  
The Stability

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH2-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH2-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH2-DA in cell culture medium relied on medium composition. The use of DCFH2-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

‘Transdifferentiation’ of chicken neural retina into lens and pigment epithelium in culture: controlling influences

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 1-21
Author(s):  
D. J. Pritchard ◽  
R. M. Clayton ◽  
D. I. De Pomerai
Keyword(s):  
Pigment Cell ◽  
Pigment Epithelium ◽  
Neural Retina ◽  
Medium Composition ◽  
Pigment Cells ◽  
Bicarbonate Concentration ◽  
Experimental Conditions ◽  
Culture Growth ◽  
New Hypothesis

The in vitro transdifferentiation of chicken embryo neural retina into pigment epithelium and lens cells was investigated under a variety of experimental conditions. Our findings suggest that some aspects of the phenomena are a function of medium composition and volume, whereas others depend upon conditions which develop during culture growth. Before melanin is visible, potential pigment cells are recognized as foci within epithelialsheets which remain in contact with the dish. The final area occupied by colonies of potential pigment cells is directly proportional to bicarbonate concentration. Low total medium volume also favours formation of potential pigment cells. In contrast the extent of cells other than potential pigment cells is not related to bicarbonate and is favoured when the volume of medium is large. Accumulation of melanin within the potential pigment cell colonies is suppressed when cells are crowded together. Lentoid bodies are formed from cells which are distinct from potential pigment cells and arise in crowded situations, in association with multilayering. Another type of structure superficially resembling a lentoid is derived from cell aggregates formed during the initial establishment of cultures. The survival of these ‘aggregate bodies’ is inversely related to bicarbonate concentration. Crystallin content is unrelated to lentoid numbers. The results provide the basis for a new hypothesis concerning cytodifferentiation in this system.

scholarly journals In vitro biological assessment of the stability of cigarette smoke aqueous aerosol extracts

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Mark Taylor ◽  
Simone Santopietro ◽  
Andrew Baxter ◽  
Nicole East ◽  
Damien Breheny ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cigarette Smoke ◽  
Culture Media ◽  
Physical Stability ◽  
Bronchial Epithelial Cell ◽  
Biological Assessment ◽  
Gas Chromatography Mass Spectrometry ◽  
Fresh Sample ◽  
The Stability

Abstract Objectives Cigarette smoke aqueous aerosol extracts (AqE) have been used for assessing tobacco products, particularly with in vitro models such as oxidative stress and inflammation. These test articles can be generated easily, but there are no standardised methods for the generation and characterisation or stability. We investigated the effects of pro-oxidant smoke-derived chemicals by using 3R4F AqE generated under standardised conditioning and smoking regimes and assessed the stability over 31-week timeframe. Twenty batches generated from ten puffs per cigarette bubbled through 20 ml cell culture media were used fresh and thawed from frozen aliquots stored at – 80 ºC. Results Nicotine levels quantified by gas chromatography/mass spectrometry and optical density at 260 nm showed chemical and physical stability from week 0 (fresh sample) to weeks 1, 4, 8 and 31 (frozen samples). No significant change in H292 human bronchial epithelial cell viability or oxidative stress were observed between fresh AqE at week 0 and frozen AqE at 31 weeks. AqEs generated by our protocol were stable for up to 31 weeks for all tested end points, suggesting that it may not be necessary to use freshly generated AqE for each study, thus reducing batch-to-batch variability.

The Influence of Experimental Conditions on the Preparation of 99mTc-Pyrrolidino-Methyl Tetracycline [99mTc-PMT] in the Form of an Instant Kit

1988 ◽  
Vol 27 (04) ◽  
pp. 154-156
Author(s):  
M. H. S. Al-Hissoni ◽  
M. H. Jassim ◽  
Y. F. Shafiq
Keyword(s):  
Organ Distribution ◽  
Experimental Conditions ◽  
Renal Scanning ◽  
The Stability

The effects of varying the experimental conditions of the preparation of the renal scanning agent 99mTc-PMT on its organ distribution have been investigated. The stability of 99mTc-PMT was determined in vitro. A procedure for the preparation of this agent is recommended.

scholarly journals Zn2+ Interaction with Amyloid-Β: Affinity and Speciation

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2796 ◽  
Author(s):  
Arena ◽  
Rizzarelli
Keyword(s):  
Species Distribution ◽  
Multiple Equilibria ◽  
Amyloid Β ◽  
Data Interpretation ◽  
Amyloid Β Peptide ◽  
Experimental Conditions ◽  
Pros And Cons ◽  
The Stability ◽  
Ph Range

Conflicting values, obtained by different techniques and often under different experimental conditions have been reported on the affinity of Zn2+ for amyloid-β, that is recognized as the major interaction responsible for Alzheimer’s disease. Here, we compare the approaches employed so far, i.e., the evaluation of Kd and the determination of the stability constants to quantitatively express the affinity of Zn2+ for the amyloid-β peptide, evidencing the pros and cons of the two approaches. We also comment on the different techniques and conditions employed that may lead to divergent data. Through the analysis of the species distribution obtained for two selected examples, we show the implications that the speciation, based on stoichiometric constants rather than on Kd, may have on data interpretation. The paper also demonstrates that the problem is further complicated by the occurrence of multiple equilibria over a relatively narrow pH range.

scholarly journals Sperm Oxidative Stress Is Detrimental to Embryo Development: A Dose-Dependent Study Model and a New and More Sensitive Oxidative Status Evaluation

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Letícia S. de Castro ◽  
Patrícia M. de Assis ◽  
Adriano F. P. Siqueira ◽  
Thais R. S. Hamilton ◽  
Camilla M. Mendes ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
System Analysis ◽  
Oxidative Status ◽  
Mitochondrial Potential ◽  
Dichlorofluorescein Diacetate ◽  
Dependent Model ◽  
The Impact ◽  
Dose Dependent

Our study aimed to assess the impact of sperm oxidative stress on embryo development by means of a dose-dependent model. In experiment 1, straws from five bulls were subjected to incubation with increasing H2O2doses (0, 12.5, 25, and 50 μM). Motility parameters were evaluated by Computed Assisted System Analysis (CASA). Experiment 2 was designed to study a high (50 μM) and low dose (12.5 μM) of H2O2compared to a control (0 μM). Samples were incubated and further used forin vitrofertilization. Analyses of motility (CASA), oxidative status (CellROX green and 2’-7’ dichlorofluorescein diacetate), mitochondrial potential (JC-1), chromatin integrity (AO), and sperm capacitation status (chlortetracycline) were performed. Embryos were evaluated based on fast cleavage (30 h.p.i.), cleavage (D=3), development (D=5), and blastocyst rates (D=8). We observed a dose-dependent deleterious effect of H2O2on motility and increase on the percentages of positive cells for CellROX green, capacitated sperm, and AO. A decrease on cleavage and blastocyst rates was observed as H2O2increased. Also, we detected a blockage on embryo development. We concluded that sperm when exposed to oxidative environment presents impaired motility traits, prooxidative status, and premature capacitation; such alterations resulting in embryo development fail.

scholarly journals NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

2018 ◽  
Vol 38 (12) ◽  
Author(s):  
Sarah C. Williams ◽  
Jason L. Parsons
Keyword(s):  
Oxidative Stress ◽  
Cellular Response ◽  
Cultured Cells ◽  
Excision Repair ◽  
Thymine Glycol ◽  
Endonuclease Iii ◽  
Hydrogen Peroxide Treatment ◽  
Base Excision ◽  
The Stability

ABSTRACT Endonuclease III-like protein 1 (NTH1) is a DNA glycosylase required for the repair of oxidized bases, such as thymine glycol, within the base excision repair pathway. We examined regulation of NTH1 protein by the ubiquitin proteasome pathway and identified the E3 ubiquitin ligase tripartite motif 26 (TRIM26) as the major enzyme targeting NTH1 for polyubiquitylation. We demonstrate that TRIM26 catalyzes ubiquitylation of NTH1 predominantly on lysine 67 present within the N terminus of the protein in vitro . In addition, the stability of a ubiquitylation-deficient protein mutant of NTH1 (lysine to arginine) at this specific residue was significantly increased in comparison to the wild-type protein when transiently expressed in cultured cells. We also demonstrate that cellular NTH1 protein is induced in response to oxidative stress following hydrogen peroxide treatment of cells and that accumulation of NTH1 on chromatin is exacerbated in the absence of TRIM26 through small interfering RNA (siRNA) depletion. Stabilization of NTH1 following TRIM26 siRNA also causes significant acceleration in the kinetics of DNA damage repair and cellular resistance to oxidative stress, which can be recapitulated by moderate overexpression of NTH1. This demonstrates the importance of TRIM26 in regulating the cellular levels of NTH1, particularly under conditions of oxidative stress.

An In Vitro Toxicity Assay for Human Endothelial Cells

1988 ◽  
Vol 16 (1) ◽  
pp. 38-41
Author(s):  
Rosella Sbarbati ◽  
Maria Luisa Schinetti ◽  
Maria Scarlattini
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Diseases ◽  
In Vitro Toxicity ◽  
Human Endothelial Cells ◽  
Experimental Conditions ◽  
Umbilical Vein Endothelial Cells

Cultured human endothelial cells can replace living animals in studying the toxic role of noxious agents in the pathogenesis of vascular diseases and in the elucidation of the mechanism of action of protective drugs. Preliminary data are presented which examine the effects that oxidative stress produces on human endothelial cells in vitro. Human umbilical vein endothelial cells were subjected to an anoxia-re-oxygenation treatment and tested for the production of Super Oxide Dismutase (SOD)-inhibitable superoxide radicals. The results show that under our experimental conditions endothelial cells produce oxygen-free radicals and that the generation reaches a maximum after an anoxic challenge of 20 minutes. We conclude that the in vitro system presented in this paper could be a suitable tool for further studies on the effects of oxidative stress on the vascular endothelium, which mimics the in vivo conditions of re-perfusion after heart ischemia.

scholarly journals Co-Loading of Ascorbic Acid and Tocopherol in Eudragit-Nutriosomes to Counteract Intestinal Oxidative Stress

Pharmaceutics ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 13 ◽  
Author(s):  
Maryam Rezvani ◽  
Maria Letizia Manca ◽  
Carla Caddeo ◽  
Elvira Escribano-Ferrer ◽  
Claudia Carbone ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ascorbic Acid ◽  
Protective Effect ◽  
Natural Antioxidants ◽  
Water Soluble ◽  
Proliferative Effect ◽  
The Stability ◽  
Stress Damage ◽  
Negative Zeta Potential

The present study aimed at developing a new vesicular formulation capable of promoting the protective effect of ascorbic acid and tocopherol against intestinal oxidative stress damage, and their efficacy in intestinal wound healing upon oral administration. A pH-dependent copolymer (Eudragit® L100), a water-soluble prebiotic fibre (Nutriose® FM06), a phospholipid mixture (Lipoid S75), and two natural antioxidants (ascorbic acid and tocopherol) were combined to fabricate eudragit-nutriosomes by a simple, solvent-free procedure. The vesicles were spherical and oligolamellar, with some multicompartment structures in Eudragit-nutriosomes, small in size (~100 nm), with highly negative zeta potential. The effect of Eudragit® and Nutriose® on the stability on storage and in simulated gastrointestinal fluids were confirmed by the Turbiscan® technology and in vitro studies, respectively. Eudragit-nutriosomes exhibited a protective effect against H2O2-induced oxidative stress, and a proliferative effect in Caco-2 cells, as they provided the closure of the scratched area after 96 h of incubation.

scholarly journals Characterization of the ECB Binding Complex Responsible for the M/G1-Specific Transcription of CLN3 and SWI4

2002 ◽  
Vol 22 (2) ◽  
pp. 430-441 ◽  
Author(s):  
Bernard Mai ◽  
Shawna Miles ◽  
Linda L. Breeden
Keyword(s):  
Cell Cycle ◽  
Gel Filtration ◽  
Specific Cell ◽  
Dnase I Footprinting ◽  
Flanking Sequences ◽  
Additional Protein ◽  
Cell Type Specific ◽  
The Stability

ABSTRACT The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes. In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G1 phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB). Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein. Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences. These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element. Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes.

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