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The Journal of Physiological Sciences :... Jul 2018Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of...
Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of human TRPA1 (hTRPA1) to respond to physical stimulus, and evaluated the response of hTRPA1 to cell swelling under hypotonic conditions. Using a Ca-imaging method, we found that modulation of AITC-induced hTRPA1 activity occurred under hypotonic conditions. Moreover, cell swelling in hypotonic conditions evoked single-channel activation of hTRPA1 in a cell-attached mode when the patch pipette was attached after cell swelling under hypotonic conditions, but not before swelling. Single-channel currents activated by cell swelling were also inhibited by a known hTRPA1 blocker. Since pre-application of thapsigargin or pretreatment with the calcium chelator BAPTA did not affect the single-channel activation induced by cell swelling, changes in intracellular calcium concentrations are likely not related to hTRPA1 activation induced by physical stimuli.
Topics: Calcium; Cell Enlargement; HEK293 Cells; Humans; Hypotonic Solutions; Patch-Clamp Techniques; TRPA1 Cation Channel
PubMed: 28623463
DOI: 10.1007/s12576-017-0545-9 -
BMC Biotechnology Aug 2023The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by...
BACKGROUND
The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by mechanically removing cells through manual scraping with a sharp-edged tool. This step is traditionally done with pipette tips and is unsuitable for high-throughput assays, as the created scratches are highly variable in width and position. Commercially available solutions are often expensive, and require specific cultureware which might not be suitable for all studies.
RESULTS
In this study, we have developed a flexible cell scratch device comprising a single wounding tool, a guide and an imaging template for consistent and reproducible scratch assays in 96-well plates. Our results showed that the device produced a more consistent scratch profile compared to the conventional method of using pipette tips. The imaging template also allowed operators to easily locate and image the same region of interest at different time points, which potentially could be used for other assays.
CONCLUSIONS
Our flexible yet effective scratch device thus enables robust scratch assays that can be applied to different experimental needs, providing researchers with an easy and reliable tool for their studies.
Topics: Research Design; Biological Assay; High-Throughput Screening Assays; Wound Healing
PubMed: 37641063
DOI: 10.1186/s12896-023-00806-5 -
Journal of Research of the National... 2017
PubMed: 34877090
DOI: 10.6028/jres.122.002 -
Experimental Physiology May 2017Although patch pipettes were initially designed to record extracellularly the elementary current events from muscle and neuron membranes, the whole-cell and loose... (Review)
Review
Although patch pipettes were initially designed to record extracellularly the elementary current events from muscle and neuron membranes, the whole-cell and loose cell-attached recording configurations proved to be useful tools for examination of signalling within and between nerve cells. In this Paton Prize Lecture, I will initially summarize work on electrical signalling within single neurons, describing communication between the dendritic compartments, soma and nerve terminals via forward- and backward-propagating action potentials. The newly discovered dendritic excitability endows neurons with the capacity for coincidence detection of spatially separated subthreshold inputs. When these are occurring during a time window of tens of milliseconds, this information is broadcast to other cells by the initiation of bursts of action potentials (AP bursts). The occurrence of AP bursts critically impacts signalling between neurons that are controlled by target-cell-specific transmitter release mechanisms at downstream synapses even in different terminals of the same neuron. This can, in turn, induce mechanisms that underly synaptic plasticity when AP bursts occur within a short time window, both presynaptically in terminals and postsynaptically in dendrites. A fundamental question that arises from these findings is: 'what are the possible functions of active dendritic excitability with respect to network dynamics in the intact cortex of behaving animals?' To answer this question, I highlight in this review the functional and anatomical architectures of an average cortical column in the vibrissal (whisker) field of the somatosensory cortex (vS1), with an emphasis on the functions of layer 5 thick-tufted cells (L5tt) embedded in this structure. Sensory-evoked synaptic and action potential responses of these major cortical output neurons are compared with responses in the afferent pathway, viz. the neurons in primary somatosensory thalamus and in one of their efferent targets, the secondary somatosensory thalamus. Coincidence-detection mechanisms appear to be implemented in vivo as judged from the occurrence of AP bursts. Three-dimensional reconstructions of anatomical projections suggest that inputs of several combinations of thalamocortical projections and intra- and transcolumnar connections, specifically those from infragranular layers, could trigger active dendritic mechanisms that generate AP bursts. Finally, recordings from target cells of a column reveal the importance of AP bursts for signal transfer to these cells. The observations lead to the hypothesis that in vS1 cortex, the sensory afferent sensory code is transformed, at least in part, from a rate to an interval (burst) code that broadcasts the occurrence of whisker touch to different targets of L5tt cells. In addition, the occurrence of pre- and postsynaptic AP bursts may, in the long run, alter touch representation in cortex.
Topics: Action Potentials; Afferent Pathways; Animals; Dendrites; Excitatory Postsynaptic Potentials; Neurons; Somatosensory Cortex; Synapses; Synaptic Transmission
PubMed: 28139019
DOI: 10.1113/EP085776 -
Nature May 2019A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research....
A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research. Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo. Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour. Fig. 1 PHOTOACTIVATED QUASAR3 (PAQUASAR3) REPORTS NEURONAL ACTIVITY IN VIVO.: a, Schematic of the paQuasAr3 construct. b, Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n = 4 cells). c, Model of the photocycle of paQuasAr3. d, Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 μm. Representative images, experiments were repeated in n = 3 mice. e, Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f, Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2-3 neuron induced by electrical stimulation of L5-6 in acute slice. g, Normalized change in fluorescence (ΔF/F) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was ΔF/F = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth (n = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h, Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n = 7 CA1 cells and n = 13 olfactory bulb cells, n = 3 mice). Schematics show microscope objective and the imaged brain region. i, STA fluorescence from 88 spikes in a CA1 oriens neuron. j, Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k, Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k-m were repeated in n = 2 cells from n = 2 mice.
Topics: Action Potentials; Algorithms; Animals; Archaeal Proteins; Bacteriorhodopsins; Cells, Cultured; Female; HEK293 Cells; Hippocampus; Humans; Male; Mice; Mice, Inbred C57BL; Neurons; Optogenetics; Walking
PubMed: 31043747
DOI: 10.1038/s41586-019-1166-7 -
Scientific Reports Jan 2023Droplet microfluidics offers a platform from which new digital molecular assay, disease screening, wound healing and material synthesis technologies have been proposed....
Droplet microfluidics offers a platform from which new digital molecular assay, disease screening, wound healing and material synthesis technologies have been proposed. However, the current commercial droplet generation, assembly and imaging technologies are too expensive and rigid to permit rapid and broad-range tuning of droplet features/cargoes. This rapid prototyping bottleneck has limited further expansion of its application. Herein, an inexpensive home-made pipette droplet microfluidics kit is introduced. This kit includes elliptical pipette tips that can be fabricated with a simple DIY (Do-It-Yourself) tool, a unique tape-based or 3D printed shallow-center imaging chip that allows rapid monolayer droplet assembly/immobilization and imaging with a smart-phone camera or miniature microscope. The droplets are generated by manual or automatic pipetting without expensive and lab-bound microfluidic pumps. The droplet size and fluid viscosity/surface tension can be varied significantly because of our particular droplet generation, assembly and imaging designs. The versatility of this rapid prototyping kit is demonstrated with three representative applications that can benefit from a droplet microfluidic platform: (1) Droplets as microreactors for PCR reaction with reverse transcription to detect and quantify target RNAs. (2) Droplets as microcompartments for spirulina culturing and the optical color/turbidity changes in droplets with spirulina confirm successful photosynthetic culturing. (3) Droplets as templates/molds for controlled synthesis of gold-capped polyacrylamide/gold composite Janus microgels. The easily fabricated and user-friendly portable kit is hence ideally suited for design, training and educational labs.
Topics: Microfluidics; Microfluidic Analytical Techniques; Microgels; Cell Encapsulation; Polymerase Chain Reaction
PubMed: 36604528
DOI: 10.1038/s41598-023-27470-1 -
Molecules (Basel, Switzerland) Jun 2023This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based... (Review)
Review
This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based materials are promising for analytical chemistry, including applications as sorbents in liquid chromatography. These sorbents can be functionalized to produce unique extraction or stationary phases. Additionally, graphene-based sorbents can be supported in various materials and have consequently been applied to produce various devices for sample preparation. Graphene-based sorbents are employed in diverse applications, including food and environmental LC analysis. This review summarizes the application of graphene-based materials in food and environmental water analysis in the last five years (2019 to 2023). Offline and online sample preparation methods, such as dispersive solid phase microextraction, stir bar sorptive extraction, pipette tip solid phase extraction, in-tube solid-phase microextraction, and others, are reviewed. The review also summarizes the application of the columns produced with graphene-based materials in separating food and water components and contaminants. Graphene-based materials have been reported as stationary phases for LC columns. Graphene-based stationary phases have been reported in packed, monolithic, and open tubular columns and have been used in LC and capillary electrochromatography modes.
Topics: Graphite; Chromatography, Liquid; Solid Phase Extraction; Solid Phase Microextraction; Water
PubMed: 37446796
DOI: 10.3390/molecules28135134 -
Clinical Microbiology Newsletter Apr 2022Maybe 2021 wasn't so bad after all! It was like 2020, but with COVID vaccines and better access to plastic pipette tips, and clearly, 2022 is getting off to a bad start....
Maybe 2021 wasn't so bad after all! It was like 2020, but with COVID vaccines and better access to plastic pipette tips, and clearly, 2022 is getting off to a bad start. Even better, 2021 had some exciting papers in clinical microbiology, which is what is highlighted in this 2021 year in review. None of them are about COVID-19. Of course, there were some interesting discoveries about SARS-CoV-2 and COVID-19, but this review is completely COVID-19 free (almost).
PubMed: 35529099
DOI: 10.1016/j.clinmicnews.2022.04.003 -
Frontiers in Molecular Neuroscience 2022Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated...
Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-μM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC and IC determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.
PubMed: 36340694
DOI: 10.3389/fnmol.2022.982689 -
Proceedings. Mathematical, Physical,... Mar 2017Along with more prevalent solid-state nanopores, glass or quartz nanopipettes have found applications in resistive-pulse and rectification sensing. Their advantages... (Review)
Review
Along with more prevalent solid-state nanopores, glass or quartz nanopipettes have found applications in resistive-pulse and rectification sensing. Their advantages include the ease of fabrication, small physical size and needle-like geometry, rendering them useful for local measurements in small spaces and delivery of nanoparticles/biomolecules. Carbon nanopipettes fabricated by depositing a thin carbon layer on the inner wall of a quartz pipette provide additional means for detecting electroactive species and fine-tuning the current rectification properties. In this paper, we discuss the fundamentals of resistive-pulse sensing with nanopipettes and our recent studies of current rectification in carbon pipettes.
PubMed: 28413354
DOI: 10.1098/rspa.2016.0931