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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 -
Small Methods Nov 2023Organic semiconductors are a family of pi-conjugated compounds used in many applications, such as displays, bioelectronics, and thermoelectrics. However, their...
Organic semiconductors are a family of pi-conjugated compounds used in many applications, such as displays, bioelectronics, and thermoelectrics. However, their susceptibility to processing-induced contamination is not well understood. Here, it is shown that many organic electronic devices reported so far may have been unintentionally contaminated, thus affecting their performance, water uptake, and thin film properties. Nuclear magnetic resonance spectroscopy is used to detect and quantify contaminants originating from the glovebox atmosphere and common laboratory consumables used during device fabrication. Importantly, this in-depth understanding of the sources of contamination allows the establishment of clean fabrication protocols, and the fabrication of organic field effect transistors (OFETs) with improved performance and stability. This study highlights the role of unintentional contaminants in organic electronic devices, and demonstrates that certain stringent processing conditions need to be met to avoid scientific misinterpretation, ensure device reproducibility, and facilitate performance stability. The experimental procedures and conditions used herein are typical of those used by many groups in the field of solution-processed organic semiconductors. Therefore, the insights gained into the effects of contamination are likely to be broadly applicable to studies, not just of OFETs, but also of other devices based on these materials.
PubMed: 37661594
DOI: 10.1002/smtd.202300476 -
Advanced Materials (Deerfield Beach,... Nov 2023Electronically controlled droplet manipulation has widespread applications in biochemistry, life sciences, and industry. However, current technologies such as...
Electronically controlled droplet manipulation has widespread applications in biochemistry, life sciences, and industry. However, current technologies such as electrowetting, electrostatics, and surface charge printing rely on complex electrode arrays and external power supplies, leading to inefficient manipulation. In light of these limitations, a novel method is proposed, which leverages tribo-electrophoresis (TEP) to pipette in an oil medium, thereby enabling human-droplet interactions to be constructed with greater efficiency. The approach involves the rational design of a triboelectric nanogenerator-electrostatic tweezer that generates an electric field to charge the droplet and improves the maneuverability of the charged droplet, including aligned/non-aligned pipetting and stable transport in the clamped state, which can be accomplished solely by hand motion. The TEP method not only provides droplets with freedom to move in three dimensions but also offers a feasibility case for chemical reactions in the liquid phase and non-invasive sample extraction. This breakthrough establishes a cornerstone for human-droplet interactions capitalized on triboelectric nanogenerators, opening new avenues for research in droplet manipulation.
PubMed: 37477978
DOI: 10.1002/adma.202305578 -
Bio-protocol Nov 2023Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of...
Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of propagation velocity using a combination of somatic whole cell and axonal loose patch recordings in brain slice preparations. The axons of neurons filled with fluorescent dye via somatic whole-cell pipette can be targeted under direct optical control using the fluorophore-filled pipette. The propagation delays between the soma and 5-7 axonal locations can be obtained by analyzing the ensemble averages of 500-600 sweeps of somatic APs aligned at times of maximal rate-of-rise (dV/dtmax) and axonal action currents from these locations. By plotting the propagation delays against the distance, the location of the AP initiation zone becomes evident as the site exhibiting the greatest delay relative to the soma. Performing linear fitting of the delays obtained from sites both proximal and distal from the trigger zone allows the determination of the velocities of AP backward and forward propagation, respectively. Key features • Ultra-thin axons in cortical slices are targeted under direct optical control using the SBFI-filled pipette. • Dual somatic whole cell and axonal loose patch recordings from 5-7 axonal locations. • Ensemble averaging of 500-600 sweeps of somatic APs and axonal action currents. • Plotting the propagation delays against the distance enables the determination of the trigger zone's position and velocities of AP backward and forward propagation.
PubMed: 37969753
DOI: 10.21769/BioProtoc.4876 -
Plant Disease Oct 2023The pine pitch canker pathogen is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable...
The pine pitch canker pathogen is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to . Using a globally representative collection of isolates and other closely related species, we have demonstrated that the assay can be used to identify across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.
Topics: Fusarium; Trees; DNA
PubMed: 36867583
DOI: 10.1094/PDIS-04-22-0972-SR -
Angewandte Chemie (International Ed. in... Aug 2023"The biggest change in my scientific working environment in the past 10 years has been the transition from being gloved up with a pipette in hand to being plugged in...
"The biggest change in my scientific working environment in the past 10 years has been the transition from being gloved up with a pipette in hand to being plugged in with a laptop … The most important quality of a role model is self-awareness-knowing one's strengths and weaknesses, as no one is a finished product. One can continue to learn and grow …" Find out more about Sheel C. Dodani in her Introducing … Profile.
PubMed: 37341254
DOI: 10.1002/anie.202306838 -
American Journal of Physiology. Cell... Dec 2023Epithelial tissues form selective barriers to ions, nutrients, waste products, and infectious agents throughout the body. Damage to these barriers is associated with...
Epithelial tissues form selective barriers to ions, nutrients, waste products, and infectious agents throughout the body. Damage to these barriers is associated with conditions such as celiac disease, cystic fibrosis, diabetes, and age-related macular degeneration. Conventional electrophysiology measurements like transepithelial resistance can quantify epithelial tissue maturity and barrier integrity but are limited in differentiating between apical, basolateral, and paracellular transport pathways. To overcome this limitation, a combination of mathematical modeling, stem cell biology, and cell physiology led to the development of 3 P-EIS, a novel mathematical model and measurement technique. 3 P-EIS employs an intracellular pipette and extracellular electrochemical impedance spectroscopy to accurately measure membrane-specific properties of epithelia, without the constraints of prior models. 3 P-EIS was validated using electronic circuit models of epithelia with known resistances and capacitances, confirming a median error of 19% (interquartile range: 14%-26%) for paracellular and transcellular resistances and capacitances ( = 5). Patient stem cell-derived retinal pigment epithelium tissues were measured using 3 P-EIS, successfully isolating the cellular responses to adenosine triphosphate. 3 P-EIS enhances quality control in epithelial cell therapies and has extensive applicability in drug testing and disease modeling, marking a significant advance in epithelial physiology. This interdisciplinary paper integrates mathematics, biology, and physiology to measure epithelial tissue's apical, basolateral, and paracellular transport pathways. A key advancement is the inclusion of intracellular voltage recordings using a sharp pipette, enabling precise quantification of relative impedance changes between apical and basolateral membranes. This enhanced electrochemical impedance spectroscopy technique offers insights into epithelial transport dynamics, advancing disease understanding, drug interactions, and cell therapies. Its broad applicability contributes significantly to epithelial physiology research.
Topics: Humans; Epithelium; Epithelial Cells; Retinal Pigment Epithelium; Cell Membrane; Models, Theoretical
PubMed: 37899750
DOI: 10.1152/ajpcell.00200.2023 -
Light, Science & Applications Aug 2023The dynamics and structure of mixed phases in a complex fluid can significantly impact its material properties, such as viscoelasticity. Small-angle X-ray Photon...
The dynamics and structure of mixed phases in a complex fluid can significantly impact its material properties, such as viscoelasticity. Small-angle X-ray Photon Correlation Spectroscopy (SA-XPCS) can probe the spontaneous spatial fluctuations of the mixed phases under various in situ environments over wide spatiotemporal ranges (10-10 s /10-10 m). Tailored material design, however, requires searching through a massive number of sample compositions and experimental parameters, which is beyond the bandwidth of the current coherent X-ray beamline. Using 3.7-μs-resolved XPCS synchronized with the clock frequency at the Advanced Photon Source, we demonstrated the consistency between the Brownian dynamics of ~100 nm diameter colloidal silica nanoparticles measured from an enclosed pendant drop and a sealed capillary. The electronic pipette can also be mounted on a robotic arm to access different stock solutions and create complex fluids with highly-repeatable and precisely controlled composition profiles. This closed-loop, AI-executable protocol is applicable to light scattering techniques regardless of the light wavelength and optical coherence, and is a first step towards high-throughput, autonomous material discovery.
PubMed: 37596264
DOI: 10.1038/s41377-023-01233-z -
Journal of Chromatography. A Sep 2023In this study, the packed sorbent solid phase (micro) extraction methods from manual to automated modes are reviewed. The automatic methods have several remarkable... (Review)
Review
In this study, the packed sorbent solid phase (micro) extraction methods from manual to automated modes are reviewed. The automatic methods have several remarkable advantages such as high sample throughput, reproducibility, sensitivity, and extraction efficiency. These methods include solid-phase extraction, pipette tip micro-solid phase extraction, microextraction by packed sorbent, in-tip solid phase microextraction, in-tube solid phase microextraction, lab-on-a-chip, and lab-on-a-valve. The recent application of these methods for the extraction of organic and inorganic compounds are discussed. Also, the combination of novel technologies (3D printing and robotic platforms) with the (semi)automated methods are investigated as the future trend.
Topics: Environmental Pollutants; Reproducibility of Results; Solid Phase Extraction; Solid Phase Microextraction
PubMed: 37506462
DOI: 10.1016/j.chroma.2023.464227 -
Cryo Letters 2023Amides are low molecular weight cryoprotectants. N-methylacetamide (MA) is one of the cryoprotectant agents in this group.
BACKGROUND
Amides are low molecular weight cryoprotectants. N-methylacetamide (MA) is one of the cryoprotectant agents in this group.
OBJECTIVE
To investigate the cryoprotective effect of MA in rabbit semen.
MATERIALS AND METHODS
For this purpose, six ejaculates from six New Zealand rabbits were collected and pooled using an artificial vagina. Pooled semen was divided into four equal parts and diluted with TCG+ egg yolk. CPA was added to form the following groups: Control with 6% DMSO; Group 1 with 1% MA; Group 2 with 2% MA; and Group 3 with 3% MA. After the addition of CPA, the semen eqilibration procedure was started. Sperm were then drawn into 0.25 mL straws, frozen by automatic semen freezing and stored in a liquid nitrogen container. Pipettes were thawed after 24 h and analyses were performed.
RESULTS
Total, progressive and rapid motility values of the Control group were higher than those of the MA groups (p<0.05). However, there was no statistical difference between the Control and Group 2 in terms of these parameters. While there was no statistical difference between the groups in terms of acrosome damage and mitochondrial membrane potential, the best results were observed in Control, Group 2, Group 1 and Group 3, respectively. When we compared all groups, no difference was found in terms of MDA, CAT and GSH-Px. There was a statistical difference between Group 3 and the Control in terms of GSH level (p<0.05).
CONCLUSION
DMSO appeared to be more useful for the cryopreservation of rabbit semen compared to MA. Doi.org/10.54680/fr23610110812.
Topics: Female; Rabbits; Male; Animals; Cryoprotective Agents; Semen; Dimethyl Sulfoxide; Cryopreservation; Semen Preservation; Sperm Motility; Spermatozoa; Semen Analysis; Acetamides
PubMed: 38311932
DOI: No ID Found