scholarly journals Experimental Conditions and Mathematical Analysis of Kinetic Measurements Using Flow Cytometry – The FacsKin Method

10.5772/38513 ◽  
2012 ◽  
Author(s):  
Ambrus Kaposi ◽  
Gergely Toldi ◽  
Gergoo Meszaros ◽  
Balazs Szalay ◽  
Gabor Veress ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mathematical Analysis ◽  
Experimental Conditions ◽  
Kinetic Measurements

scholarly journals Investigations of ternary complexes of Co(II) and Ni(II) with thiodiacetate anion and 1,10-phenanthroline or 2,2’-bipyridine in aqueous solutions

2014 ◽  
Vol 13 (1) ◽  
Author(s):  
Dariusz Wyrzykowski ◽  
Joanna Pranczk ◽  
Dagmara Jacewicz ◽  
Aleksandra Tesmar ◽  
Bogusław Pilarski ◽  
...  
Keyword(s):  
Aqueous Solutions ◽  
Substitution Reactions ◽  
Experimental Conditions ◽  
Kinetic Measurements ◽  
Hydroxo Complexes ◽  
Binary Complexes ◽  
Vis Spectroscopy ◽  
Potentiometric Titration Method ◽  
The Stability ◽  
Ph Range

AbstractA potentiometric titration method (PT) and a stopped-flow kinetic technique monitored by a UV−Vis spectroscopy have been used to characterize the stability of series of Co(II)- and Ni(II)-thiodiacetato complexes, M(TDA), in the presence of 1,10-phenanthroline (phen) or 2,2’-bipyridine (bipy) in aqueous solutions. The stability constants of the binary (1:1), ternary (1:1:1) as well as the resulting hydroxo complexes were evaluated and compared to the corresponding oxydiacetate complexes. Based on the species distribution as a function of pH the relative predominance of the species in the system over a pH range was discussed. Furthermore, the kinetic measurements of the substitution reactions of the aqua ligands to phen or bipy in the coordination sphere of the binary complexes M(TDA) were performed in the 288–303 K temperature range, at a constant concentration of phen or bipy and at seven different concentrations of the binary complexes (0.2–0.5 mM). The kinetic stability of the M(TDA) complexes was discussed in relation to the experimental conditions and the kind of the auxiliary ligands (phen/bipy). Moreover, the influence of the type of primary ligand (thiodiacetate/oxydiacetate) on the substitution rate of the auxiliary ligands was also compared.

scholarly journals Interaction of phenylthiolato-(2,2′,2′′-terpyridine)platinum(II) cation with DNA

1984 ◽  
Vol 222 (1) ◽  
pp. 203-215 ◽  
Author(s):  
L P G Wakelin ◽  
W D McFadyen ◽  
A Walpole ◽  
I A G Roos
Keyword(s):  
Base Pair ◽  
Equilibrium Dialysis ◽  
Platinum Complexes ◽  
Nucleotide Composition ◽  
Contour Length ◽  
Ligand Molecule ◽  
Experimental Conditions ◽  
Kinetic Measurements ◽  
Binding Isotherms ◽  
Scatchard Plots

The interaction between a novel aromatic thiolato derivative from the family of DNA-intercalating platinum complexes, phenylthiolato-(2,2′,2″-terpyridine)platinum(II)-[PhS(ter py)Pt+], and nucleic acids was studied by using viscosity, equilibrium-dialysis and kinetic measurements. Viscosity measurements with sonicated DNA provide direct evidence for intercalation, and show that at binding ratios below 0.2 molecules per base-pair PhS(terpy)Pt+ causes an increase in contour length of 0.2 nm per bound molecule. However, helix extension diminishes at greater extents of binding, indicating the existence of additional, non-intercalated, externally bound forms of the ligand. The ability of PhS(terpy)Pt+ to aggregate in neutral aqueous buffers at a range of ionic strengths and temperatures was assessed by using optical-absorption methods. Scatchard plots for binding to calf thymus DNA at ionic strength 0.01 (corrected for dimerization) are curvilinear, concave upward, providing further evidence for two modes of binding. The association constant decreases at higher ionic strengths, in accord with the expectations of polyelectrolyte theory, although the number of cations released per bound unipositive ligand molecule is substantially greater than 1. Stopped-flow kinetic measurements confirm the complexity of the binding reaction by revealing multiple bound forms of the ligand whose kinetic processes are both fast and closely coupled. Thermal denaturation of DNA radically alters the shapes of binding isotherms and either has little effect on, or enhances, the affinity of potential binding sites, depending on experimental conditions. Scatchard plots for binding to natural DNA species with differing nucleotide composition show that the ligand has a requirement for a single G X C base-pair at the highest-affinity intercalation sites.

scholarly journals A call to adopt a “fit for purpose” approach to antibody validation for flow cytometry analyses of stem cell models and beyond

2019 ◽  
Vol 317 (5) ◽  
pp. H954-H957 ◽  
Author(s):  
Matthew Waas ◽  
Rebekah L. Gundry
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Published Data ◽  
Cell Models ◽  
Human Pluripotent Stem Cell ◽  
Experimental Conditions ◽  
Wide Range ◽  
Fit For Purpose ◽  
Antibody Validation ◽  
Stem Cell Models

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can be exploited as models for a wide range of research applications and numerous protocols for generating hPSC-CMs have been described. However, it is currently not possible to direct differentiation to a single, homogeneous end point, and the resulting heterogeneity may be variable among laboratories, cell lines, and protocols. Consequently, the ability to assess phenotypic heterogeneity of the cell population is critical to the interpretation, repeatability, and reproduction of hPSC-CM studies. While flow cytometry is well suited for this purpose, a review of published literature reveals there is currently no consensus regarding which marker, antibody, or protocol is best suited to enable comparisons of hPSC-CM culture heterogeneity. Moreover, the lack of available experimental detail, combined with the variability in the approaches used for hPSC-CM evaluation, makes it challenging to reproduce, interpret, and compare published data. Consequently, this article calls for an alignment of the way researchers approach the routine use and documentation of the antibodies and controls used during flow cytometry-based assessment of hPSC-CM cultures. We advocate for the adoption of a “fit for purpose” validation mindset, whereby antibodies and experimental conditions are demonstrated as specific within a defined experimental design and biological context. Overall, we expect that by adhering to rigorous standards for antibody validation and use, reporting of experimental details, and presentation of data, the concepts emphasized here will promote enhanced utility and dialogue regarding hPSC-CM for a variety of research and translational applications by enabling more accurate comparisons of results among studies. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/fit-for-purpose-approach-to-antibody-validation/ .

The measurement and prediction of sustained temperature oscillations in a chemical reactor

1969 ◽  
Vol 309 (1496) ◽  
pp. 1-26 ◽  
Keyword(s):  
Mathematical Analysis ◽  
Methyl Chloride ◽  
Chemical Reactor ◽  
Vapour Phase ◽  
Gas Temperature ◽  
Experimental Conditions ◽  
Temperature Oscillations ◽  
Series Of Experiments ◽  
Steady Oscillations

Steady oscillations in the recorded gas temperature have been observed in a series of experiments in which the vapour-phase chlorination of methyl chloride was carried out. The instability was reproducible and persisted within a sharply defined range of reaction temperatures. A mathematical analysis of the dynamics of the reacting system is found to predict closely the nature and frequency of the oscillations and the range of experimental conditions within which they occur.

Time: a new parameter for kinetic measurements in flow cytometry

Science ◽  
1980 ◽  
Vol 207 (4427) ◽  
pp. 199-201 ◽  
Author(s):  
J. Martin ◽  
D. Swartzendruber
Keyword(s):  
Flow Cytometry ◽  
Kinetic Measurements

Establishment of experimental conditions for preserving samples of fish blood for analysis with both comet assay and flow cytometry

2009 ◽  
Vol 673 (1) ◽  
pp. 78-81 ◽  
Author(s):  
Wanessa A. Ramsdorf ◽  
Fernando de S.F. Guimarães ◽  
Marcos V.M. Ferraro ◽  
Juarez Gabardo ◽  
Edvaldo da Silva Trindade ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Comet Assay ◽  
Experimental Conditions ◽  
Fish Blood

Serotoninergic Mechanisms Play a Major Role on Platelet-Mediated Thrombogenicity: Studies under Flow Conditions and Effects of Inhibitory Strategies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2636-2636
Author(s):  
Ana M. Galan ◽  
Irene Lopez-Vilches ◽  
Maribel Diaz-ricart ◽  
Fulgencio Navalon ◽  
Marcos Pino ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Serotonin Reuptake ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Annexin V ◽  
Selective Serotonin ◽  
Experimental Conditions ◽  
Factor Va ◽  
Low Concentrations ◽  
Platelet Aggregates

Abstract Serotoninergic mechanisms are reciprocally implicated in depression and cardiovascular disorders. Serotonin (5-HT) may facilitate the development of a subpopulation of platelets with increased procoagulant activity. We have investigated the involvement of serotoninergic mechanisms in adhesive, cohesive and procoagulant function of platelets. Furthermore, we evaluated the antithrombotic properties of selective serotonin reuptake inhibitors (SSRI). For these purposes we used a series of experimental strategies including standard aggregometry, flow cytometry, perfusion techniques and determination of coagulation parameters. Serotonin concentrations ranging 0.5–5 μM were used in these studies. Citalopram, a selective serotonin reuptake inhibitor (SSRI), was used at concentrations equivalent to those reached in clinical practice. Aggregation studies indicate that 5-HT is a weak agonist for platelets in comparison with standard activating agents. Only the highest concentrations of 5-HT tested (5 μM) caused minimal and reversible platelet aggregation. Despite this modest effect on aggregation, 5-HT potentiated the aggregation induced by low concentrations of ADP (0.5 μM) in PRP samples (16.3±5.9 % vs. 28.1±5.5 %). Potentiation of aggegation was more evident when PRP was obtained from blood anticoagulated with LMWH (p<0.05), where some thrombin could be generated. Citalopram at 0.6 μM caused statistical reductions in % of maximal aggregation induced by ADP (2 or 0.5 μM) and COL (2.5 μg/ml) (p<0.05). Platelet aggregation was maximally inhibited with citalopram at concentrations exceeding clinical doses (6 μM). Flow cytometry studies revealed a mild increase of P-selectin expression in washed platelets in the first minute after activation with 5-HT. In isolated platelets, 5-HT produced irreversible aggregation when studies were performed in the presence of human-TF enriched microvesicles. A significant increase in Annexin V binding and presence of factor Va was detected by flow cytometry in the latter studies. Further studies performed in perfusion models using human blood flowing though chambers exposing a surface rich in collagen and TF demonstrated that presence of 5-HT in the perfusates increased thrombin generation (p<0.05) as demonstrated by elevations of F1+2 levels. Presence of citalopram in the perfusates significantly inhibited the formation of large platelet aggregates on the perfused thrombogenic surfaces (20.2±3.4 % vs. 11.5±1.7 %; p<0.05) and prevented elevations of F1+2 levels observed in previous studies. Our studies confirm that 5-HT is a weak agonist for platelets, though it possesses a marked ability to enhance the activation induced by other agonists. Moreover, 5-HT showed a powerful capacity to potentiate procoagulant responses of platelets under different experimental conditions and to enhance the thrombogenesis on damaged vascular surfaces. The fact that SSRI inhibited the responses elicited by 5-HT in the previous situations further confirm the implications of serotoninergic mechanisms in platelet activation. Our data reinforce the opinion that serotoninergic mechanisms play an important role in platelet-mediated thrombogenicity, suggesting that modulation of 5-HT mediated responses may offer a new potential target for the development of more powerful antithrombotic drugs.

Rate constants for reactions of horseradish peroxidase compounds I and II with 4-substituted arylboronic acids

1994 ◽  
Vol 72 (10) ◽  
pp. 2159-2162 ◽  
Author(s):  
Weimei Sun ◽  
Xiaoying Ji ◽  
Larry J. Kricka ◽  
H. Brian Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Rate Constants ◽  
Compound I ◽  
Arylboronic Acid ◽  
Experimental Conditions ◽  
Kinetic Measurements ◽  
Arylboronic Acids ◽  
Total Ionic Strength ◽  
Compound Ii ◽  
Catalyzed Oxidation

The rate constants for the reactions of horseradish peroxidase compound I (k1) and compound II (k2) with three 4-substituted arylboronic acids, which enhance chemiluminescence in the horseradish peroxidase catalyzed oxidation of luminol by hydrogen peroxide, were determined at pH 8.6, total ionic strength 0.11 M, using stopped-flow kinetic measurements. For comparison, the rate constants of the reactions of 4-iodophenol with compounds I and II were also determined under the same experimental conditions. The three arylboronic acid derivatives and their rate constants are: 4-biphenylboronic acid, k1 = (1.21 ± 0.08) × 106 M−1 s−1, k2 = (4.6 ± 0.2) × 105 M−1 s−1; 4-bromophenylboronic acid, k1 = (5.5 ± 0.2) × 104 M−1 s−1, k2 = (3.6 ± 0.2) × 104 M−1 s−1; and 4-iodophenylboronic acid, k1 = (1.1 ± 0.2) × 105 M−1 s−1, k2 = (1.3 ± 0.1) × 104 M−1 s−1. 4-Biphenylboronic acid, which shows comparable luminescent enhancement to 4-iodophenol, has the highest reactivity in the reduction of both compounds I and II among the three arylboronic acid derivatives tested.

scholarly journals Application of the Micro Biological Survey analytical method for the determination of bacterial load in cow raw milk

2020 ◽  
Vol 9 (3) ◽  
Author(s):  
Alessandra Cornacchia ◽  
Maria Antonietta Saletti ◽  
Violeta Di Marzio ◽  
Romolo Salini ◽  
Cristina Marfoglia ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Bacterial Load ◽  
Colorimetric Method ◽  
Independent Set ◽  
Raw Milk ◽  
Plate Count ◽  
Experimental Conditions ◽  
Biological Survey ◽  
Plate Count Method

The aim of this study was to evaluate the performance of “Micro Biological Survey – MBS Test” in the enumeration of bacterial load in cow raw milk. The MBS test is based on a colorimetric method recently developed and patented by “Roma Tre” University, Italy. The evaluation of the performance of the MBS method was carried out by comparison with plate count at 30°C (gold standard) and flow cytometry. Thirteen independent set of experiments were performed analyzing a total of 104 samples of cow raw milk with the selected methods. Results obtained using the MBS method are comparable with those obtained with the plate count method at 30°C (CFU/mL) and flow cytometry technology; in particular, the results obtained with the MBS method are very close to plate count’s at 30°C. On the other hand, there are statistically significant differences between these two methods’ and flow cytometry technology’s results that could be due to the different experimental conditions.

scholarly journals Phagocytic activity and oxygen metabolism of peripheral blood granulocytes from rabbits experimentally infected with Trichophyton mentagrophytes

2018 ◽  
Vol 62 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Katarzyna Wojcicka-Lorenowicz ◽  
Krzysztof Kostro ◽  
Urszula Lisiecka ◽  
Bolesław Gąsiorek
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Phagocytic Activity ◽  
Spontaneous Recovery ◽  
Trichophyton Mentagrophytes ◽  
Oxygen Metabolism ◽  
Experimental Conditions ◽  
The Mean ◽  
Activity Indices

AbstractIntroductionPhagocytic activity and oxygen metabolism of peripheral blood granulocytes from rabbits with experimental trichophytosis were assessed by flow cytometry.Material and MethodsVirulent species of T. mentagrophytes var. granulosum (Tm-K) isolated from rabbits with natural trichophytosis was used for experimental infection. The phagocytic activity of granulocytes was measured in whole blood by flow cytometry using the commercial Phagotest kit. Oxidative burst was measured in whole blood by flow cytometry using the commercial Bursttest kit.ResultsIt was found that rabbits were susceptible to infection with Trichophyton mentagrophytes under experimental conditions. The analysis of the phagocytic activity indices and oxygen metabolism of granulocytes in peripheral blood of infected rabbits showed that changes of the indices were connected with the progression and regression of the disease. A significant decrease in phagocytic activity and oxygen metabolism was observed during development of fungal lesions and it remained similar throughout the progress of the disease. The highest means of the percentage of activated and ingesting phagocytes and a significant increase in the mean fluorescence intensity (representing the number of ingested bacteria) were observed during spontaneous recovery. Therefore, the decrease or increase in the indices of phagocytic activity and oxygen metabolism of granulocytes from rabbits experimentally infected with T. mentagrophytes is somehow related to the progress of infection and suppressive activity of the fungus, whose elimination during recovery caused significant increases in investigated indices of non-specific cellular immunity.ConclusionThe results of the present investigation confirm that the mechanism of oxygen-dependent killing is crucial in infections caused by T. mentagrophytes.

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