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Food Chemistry Mar 2023A simple sample preparation approach employing the dispersive pipette extraction (DPX) technique is proposed to determine twelve polyphenols, including phenolic acids...
A simple sample preparation approach employing the dispersive pipette extraction (DPX) technique is proposed to determine twelve polyphenols, including phenolic acids and flavonoids in wines, followed by identification and quantification by LC-MS/MS. The extraction parameters, including sample volume and pH, salting out effect, time and cycles of extraction and desorption, and desorption solvent were optimized using univariate and multivariate designs. The analytical performance was satisfactory, with determination coefficients greater than or equal to 0.9877, precisions with values lower than 20 %, and recoveries ranging from 87 to 114 %. The applicability of the method was evaluated in red wine. The major compounds determined in the sample were (-)-epicatechin (23.5 mg L), (+)-catechin (19.2 mg L), and myricetin (14.6 mg L). The green character of the analytical procedure and the sample preparation step were evaluated by three analytical metrics.
Topics: Chromatography, Liquid; Wine; Polyphenols; Tandem Mass Spectrometry; Solvents
PubMed: 36370557
DOI: 10.1016/j.foodchem.2022.134860 -
Journal of Neurophysiology Jul 2000The reversibility and cation selectivity of the K(+)-Cl(-) cotransporter (KCC), which normally extrudes Cl(-) out of neurons, was investigated in dissociated lateral...
The reversibility and cation selectivity of the K(+)-Cl(-) cotransporter (KCC), which normally extrudes Cl(-) out of neurons, was investigated in dissociated lateral superior olive neurons of rats using the gramicidin perforated patch technique. Intracellular Cl(-) activity (alpha[Cl(-)](i)) was maintained well below electrochemical equilibrium as determined from the extracellular Cl(-) activity and the holding potential, where the pipette and external solutions contained 150 mM K(+) ([K(+)](pipette)) and 5 mM K(+) ([K(+)](o)), respectively. Extracellular application of 1 mM furosemide or elevated [K(+)](o) increased alpha[Cl(-)](i). When the pipette solution contained 150 mM Cs(+) ([Cs(+)](pipette)), alpha[Cl(-)](i) increased to a value higher than the passive alpha[Cl(-)](i). An increase of alpha[Cl(-)](i) with the [Cs(+)](pipette) was not due to the simple blockade of net KCC by the intracellular Cs(+) since alpha[Cl(-)](i), with the pipette solution containing 75 mM Cs(+) and 75 mM K(+), reached a value between those obtained using the [K(+)](pipette) and the [Cs(+)](pipette). The higher-than-passive alpha[Cl(-)](i) with the [Cs(+)](pipette) was reduced by 1 mM furosemide, but not by 20 microM bumetanide or Na(+)-free external solution, indicating that the accumulation of [Cl(-)](i) in the [Cs(+)](pipette) was mediated by a KCC operating in a reversed mode rather than by Na(+)-dependent, bumetanide-sensitive mechanisms. Replacement of K(+) in the pipette solution with either Li(+) or Na(+) mimicked the effect of Cs(+) on alpha[Cl(-)](i). On the other hand, Rb(+) mimicked K(+) in the pipette solution. These results indicate that K(+) and Rb(+), but not Cs(+), Li(+), or Na(+), can act as substrates of KCC in LSO neurons.
Topics: Animals; Biological Transport; Bumetanide; Carrier Proteins; Cations; Cesium; Chlorides; Diuretics; Female; Furosemide; Male; Neurons; Olivary Nucleus; Patch-Clamp Techniques; Potassium; Rats; Rats, Wistar; Symporters; K Cl- Cotransporters
PubMed: 10899203
DOI: 10.1152/jn.2000.84.1.281 -
Biophysical Journal Aug 1988Study of the mechanical properties of leukocytes is useful to understand their passage through narrow capillaries and interaction with other cells. Leukocytes are known...
Study of the mechanical properties of leukocytes is useful to understand their passage through narrow capillaries and interaction with other cells. Leukocytes are known to be viscoelastic and their properties have been established by micropipette aspiration techniques. Here, the recovery of leukocytes to their normal spherical form is studied after prolonged deformation in a pipette which is large enough to permit complete entry of the leukocyte. The recovery history is characterized by the time history of the major diameter (d1) and minor diameter (d2). When the cell is removed from the pipette, it shows initially a small rapid recoil followed by a slower asymptotic recovery to the spherical shape. In the presence of cell activation and formation of pseudopods, the time history for recovery is prolonged compared with passive cell recovery. If a protopod pre-existed during the holding period, the recovery only begins when the protopod starts to retract.
Topics: Cell Movement; Elasticity; Humans; Kinetics; Leukocytes; Mathematics; Models, Biological; Viscosity
PubMed: 3207829
DOI: 10.1016/S0006-3495(88)82963-1 -
Journal of Neuroscience Methods Dec 2019Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular...
BACKGROUND
Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular degeneration feasible. However, current techniques for intracellular electrophysiology - used to validate epithelial function - are painstaking and require manual skill; limiting experimental throughput.
NEW METHOD
A five-stage algorithm, leveraging advances in automated patch clamping, systematically derived and optimized, improves yield and reduces skill when compared to conventional, manual techniques.
RESULTS
The automated algorithm improves yield per attempt from 17% (manually, n = 23) to 22% (automated, n = 120) (chi-squared, p = 0.004). Specifically for RPE, depressing the local cell membrane by 6 μm and electroporating (buzzing) just prior to this depth (5 μm) maximized yield.
COMPARISON WITH EXISTING METHOD
Conventionally, intracellular epithelial electrophysiology is performed by manually lowering a pipette with a micromanipulator, blindly, towards a monolayer of cells and spontaneously stopping when the magnitude of the instantaneous measured membrane potential decreased below a predetermined threshold. The new method automatically measures the pipette tip resistance during the descent, detects the cell surface, indents the cell membrane, and briefly buzzes to electroporate the membrane while descending, overall achieving a higher yield than conventional methods.
CONCLUSIONS
This paper presents an algorithm for high-yield, automated intracellular electrophysiology in epithelia; optimized for human RPE. Automation reduces required user skill and training while, simultaneously, improving yield. This algorithm could enable large-scale exploration of drug toxicity and physiological function verification for numerous kinds of epithelia.
Topics: Algorithms; Electrophysiology; Humans; Patch-Clamp Techniques; Retinal Pigment Epithelium
PubMed: 31562888
DOI: 10.1016/j.jneumeth.2019.108442 -
RSC Advances Dec 2019Microfluidics offers great potential for biomedical applications, but the complexity, inconvenience, and low pumping equipment accessibility of operating microfluidic...
Microfluidics offers great potential for biomedical applications, but the complexity, inconvenience, and low pumping equipment accessibility of operating microfluidic devices have limited their widespread use. Here we describe an open-source, programmable smart (OS) pipette as an easy-to-use, simple, handheld microfluidic pump that overcomes the major limitations of previous commercial- or research-level pumps for microfluidics. The OS pipette pumps fluid into a microfluidic device by precisely controlling the position of the plunger of a positive-displacement micropipette with stepper motor control. The intuitive pumping mechanism of the OS pipette enables the novel features of simple fabrication, straightforward device operation, and precise, predictable, and programmable flow-rate generation as an open-source pumping tool. Controlling the OS pipette using an open-source microcontroller board not only allows straightforward generation of constant flow rates with simple source code commands, but also permits varying flow rates to be programmed (including stepwise increase and decrease of the flow rate over time, and flow-rate pulse generation). We successfully validate the OS pipette's capabilities for portable microfluidic cell separation and counting. The OS pipette has promise as a rapidly evolving and potentially transformative pumping tool that freely allows unrestricted use, distribution, reproduction, and modification even by non-expert users, and further enables diverse usages, even beyond microfluidics.
PubMed: 35541629
DOI: 10.1039/c9ra08368e -
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi =... Aug 2021Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in...
Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (μF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.
Topics: Cell Membrane; Electric Capacitance; Membrane Potentials; Patch-Clamp Techniques; Temperature
PubMed: 34459169
DOI: 10.7507/1001-5515.202007054 -
Lab on a Chip Jan 2015Micropipette aspiration measures the mechanical properties of single cells. A traditional micropipette aspiration system requires a bulky infrastructure and has a low...
Micropipette aspiration measures the mechanical properties of single cells. A traditional micropipette aspiration system requires a bulky infrastructure and has a low throughput and limited potential for automation. We have developed a simple microfluidic device which is able to trap and apply pressure to single cells in designated aspiration arrays. By changing the volume flow rate using a syringe pump, we can accurately exert a pressure difference across the trapped cells for pipette aspiration. By examining cell deformation and protrusion length into the pipette under an optical microscope, several important cell mechanical properties, such as the cortical tension and the Young's modulus, can be measured quantitatively using automated image analysis. Using the microfluidic pipette array, the stiffness of breast cancer cells and healthy breast epithelial cells was measured and compared. Finally, we applied our device to examine the gating threshold of the mechanosensitive channel MscL expressed in mammalian cells. Together, the development of a microfluidic pipette array could enable rapid mechanophenotyping of individual cells and for mechanotransduction studies.
Topics: Biomechanical Phenomena; Cell Line, Tumor; Elastic Modulus; Equipment Design; HeLa Cells; Humans; Microfluidic Analytical Techniques; Neoplasms; Phenotype
PubMed: 25361042
DOI: 10.1039/c4lc01218f -
Neuroprotection Sep 2023Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may...
OBJECTIVE
Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may contribute to mechanical brain damage. Glass pipettes with a thin tip may significantly reduce injection-associated brain damage but require access to prohibitively expensive programmable pipette pullers. This study is to remove the economic barrier to the application of minimally invasive delivery of therapeutics to the brain, such as chemical compounds, viral vectors, and cells.
METHODS
We took advantage of the rapid development of free educational online resources and emerging low-cost 3D printers by designing an affordable pipette puller (APP) to remove the cost obstacle.
RESULTS
We showed that our APP could produce glass pipettes with a sharp tip opening down to 20 μm or less, which is sufficiently thin for the delivery of therapeutics into the brain. A pipeline from pipette pulling to brain injection using low-cost and open-source equipment was established to facilitate the application of the APP.
CONCLUSION
In the spirit of frugal science, our device may democratize glass pipette-puling and substantially promote the application of minimally invasive and precisely controlled delivery of therapeutics to the brain for finding more effective therapies of brain diseases.
PubMed: 37771648
DOI: 10.1002/nep3.20 -
Journal of Research of the National... 2017
PubMed: 34877110
DOI: 10.6028/jres.122.012 -
Scientific Reports Oct 2016Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput...
Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator.
Topics: Animals; Cells, Cultured; Detergents; Glass; HEK293 Cells; Humans; Microelectrodes; Neurons; Patch-Clamp Techniques; Rats
PubMed: 27725751
DOI: 10.1038/srep35001