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Journal of Visualized Experiments : JoVE Jul 2020In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a...
In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 µL gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.
Topics: Administration, Inhalation; Animals; Catheterization; Drug Compounding; Dry Powder Inhalers; Lung; Mice, Inbred BALB C; Particle Size; Powders; RNA, Messenger
PubMed: 32773763
DOI: 10.3791/61469 -
ACS Sensors Jun 2023More anomalous transport behaviors have been observed with the rapid progress in nanofabrication technology and characterization tools. The ions/molecules inside...
More anomalous transport behaviors have been observed with the rapid progress in nanofabrication technology and characterization tools. The ions/molecules inside nanochannels can act dramatically different from those in the bulk systems and exhibit novel mechanisms. Here, we have reported the fabrication of a nanodevice, covalent organic frameworks covered theta pipette (CTP), that combine the advantages of theta pipette (TP), nanochannels framework, and field-effect transistors (FETs) for controlling and modulating the anomalous transport. Our results show that ammonia, a weak base, causes a continuous supply of ions inside covalent organic framework (COF) nanochannels, leading to an abnormally high current depending on the ionic/molecular size and the pore size of the nanochannel. Furthermore, CTP can distinguish different concentrations of ammonia and have all of the qualities of a nanosensor.
Topics: Ammonia; Metal-Organic Frameworks; Ion Transport; Ions; Research Design
PubMed: 37245157
DOI: 10.1021/acssensors.3c00041 -
Talanta Sep 2023An efficient sample preparation based on pipette tip microextraction that can be used for the analysis of retinol in human serum has been developed. Altogether, nine...
An efficient sample preparation based on pipette tip microextraction that can be used for the analysis of retinol in human serum has been developed. Altogether, nine commercial pipette tips were compared based on recovery, sample volume, use of organic solvent, handling difficulty, duration of the preparation process, price, and greenness of the method. Retinol acetate was used as the internal standard. The extraction efficiency for both compounds was evaluated to optimize and select the best pipette tip for sample preparation, which was the WAX-S XTR pipette tip containing an ion exchanger and salt. This tip combined solid phase extraction and salting-out assisted liquid‒liquid extraction. Satisfying recoveries of 100 and 80% for retinol and retinol acetate, respectively, and good repeatability were demonstrated. The action of this pipette tip was based on the clean-up workflow in which the interferences were retained on the sorbent. The presence of residual interferences in the extracted samples did not affect the HPLC separation of compounds of interest. The simplicity of the clean-up workflow reduced the time of the sample preparation compared to the bind-wash-elute counterpart workflow. The advantages of our technique are its environmental friendliness and cost effectiveness. The selected pipette tip with an excellent microextraction efficiency enables sample preparation in both clinical research and practice.
Topics: Humans; Vitamin A; Solid Phase Extraction; Diterpenes; Retinyl Esters; Sodium Chloride
PubMed: 37220691
DOI: 10.1016/j.talanta.2023.124689 -
Faraday Discussions Apr 2022Nanoscale electrochemical mapping techniques, , scanning electrochemical cell microscopy (SECCM), have been increasingly used to study the local electrochemistry in...
Nanoscale electrochemical mapping techniques, , scanning electrochemical cell microscopy (SECCM), have been increasingly used to study the local electrochemistry in electrocatalysis. Its capability for local electrochemistry mapping helps to reveal the heterogeneity in the electrode kinetics and mechanisms, which are otherwise averaged out in ensemble measurements. Accurate determination of the electrode kinetics requires the careful assessment of the ohmic potential drop in the solution, , the drop. Herein, the drop in SECCM experiments is assessed. We showed that the drop in single-barrel SECCM can be estimated using the solution conductivity and pipette geometry, or the mass transfer limiting current without the assumption of pipette geometry. For dual-barrel SECCM, we developed a method of measuring the solution resistance directly, which can be used to compensate for the drop and the potential shift in the experiments. These methods offer a convenient way to estimate and compensate for the drop in SECCM, allowing the more accurate measurement of local electrode kinetics for the determination of local mechanisms in electrocatalysis.
Topics: Electrochemical Techniques; Electrochemistry; Electrodes; Microscopy; Microscopy, Electrochemical, Scanning
PubMed: 34877955
DOI: 10.1039/d1fd00046b -
Scientific Reports Apr 2018In order to improve the accuracy and reliability of micropipetting, a method of micro-pipette detection and calibration combining the dynamic pressure monitoring in...
In order to improve the accuracy and reliability of micropipetting, a method of micro-pipette detection and calibration combining the dynamic pressure monitoring in pipetting process and quantitative identification of pipette volume in image processing was proposed. Firstly, the normalized pressure model for the pipetting process was established with the kinematic model of the pipetting operation, and the pressure model is corrected by the experimental method. Through the pipetting process pressure and pressure of the first derivative of real-time monitoring, the use of segmentation of the double threshold method as pipetting fault evaluation criteria, and the pressure sensor data are processed by Kalman filtering, the accuracy of fault diagnosis is improved. When there is a fault, the pipette tip image is collected through the camera, extract the boundary of the liquid region by the background contrast method, and obtain the liquid volume in the tip according to the geometric characteristics of the pipette tip. The pipette deviation feedback to the automatic pipetting module and deviation correction is carried out. The titration test results show that the combination of the segmented pipetting kinematic model of the double threshold method of pressure monitoring, can effectively real-time judgment and classification of the pipette fault. The method of closed-loop adjustment of pipetting volume can effectively improve the accuracy and reliability of the pipetting system.
Topics: Calibration; Immunoenzyme Techniques; Models, Theoretical; Reproducibility of Results
PubMed: 29636540
DOI: 10.1038/s41598-018-24145-0 -
BioRxiv : the Preprint Server For... Jan 2023Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular...
Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular weight increases from 30 to 2000, roughly as expected for pores with dimensions of cardiac porin channels. The Bodipy-FL ATP analogue diffuses ∼50-fold slower in BL6 cardiac cytoplasm than in free water. From several fluorophores analyzed, our estimates of bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. We estimate that diffusion coefficients of unbound fluorophores range from 0.5 to 8 x 10 cm /s. Analysis of Na/K pump and veratridine-modified Na channel currents confirms that Na diffusion is nearly unrestricted (time constant for equilibration with the pipette tip, ∼20 s). Using three different approaches, we estimate that ATP diffuses 8 to 10-times slower in the cytoplasm of BL6 myocytes than in free water. To address whether restrictions are caused more by cytoplasmic protein or membrane networks, we verified first that a protein gel, 10 gram% gelatin, restricts solute diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is however less restricted than in intact myocytes. Notably, myofilaments from equivalently extracted skeletal (diaphragm) myocytes restrict diffusion less than cardiac myofilaments. Solute diffusion in myocytes with sarcolemma permeabilized by β-escin (80 µM) is similarly restricted as in intact myocytes. Diffusion restriction in cardiac myocytes is strain-dependent, being about two-fold greater in BL6 myocytes than in myocytes with a CD1/J6/129svJ background. Furthermore, diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, Vdac1, than in WT CD1/J6/129svJ myocytes. We conclude that both myofilaments and mitochondria networks restrict diffusion in cardiac myocytes. As a result, long-range solute diffusion may preferentially occur via passage through porin channels and intramembrane mitochondrial spaces, where diffusion is less restricted than in myofilament spaces.
PubMed: 36712045
DOI: 10.1101/2023.01.02.522313 -
Analytica Chimica Acta Feb 2022Analytical chemistry has always been evolving towards techniques that are increasingly simple and effective and that conform to green principles. Disposable pipette... (Review)
Review
Analytical chemistry has always been evolving towards techniques that are increasingly simple and effective and that conform to green principles. Disposable pipette extraction (DPX, also known as dispersive pipette tip solid phase extraction) is a recent technique that has become an interesting tool in sample preparation methodologies. The principle is based on a dynamic mixture between the matrix and the sorbent which allows rapid and effective extraction of analytes and provides vigorous clean-up of the samples. In the context of fitting in with green chemistry, DPX has contributed to replacing commercially available materials with natural alternative materials. The production of these materials is also simple, reduces sample/solvent volumes, consequently generates less waste and is less laborious and safer for the laboratory worker. This review is a source of information about the DPX technique, dealing with its basic concepts, procedure, optimizations, materials for the main applications published so far, which are in the food, environmental and biological (forensic) sciences.
Topics: Solid Phase Extraction
PubMed: 35057962
DOI: 10.1016/j.aca.2021.339383 -
Journal of Neuroscience Methods Jan 2019The composition of cerebrospinal fluid (CSF) is an invaluable parameter in better understanding of cellular and molecular processes within the mammalian brain. However,... (Review)
Review
BACKGROUND
The composition of cerebrospinal fluid (CSF) is an invaluable parameter in better understanding of cellular and molecular processes within the mammalian brain. However, the collection of significant volumes of clean CSF can be technically challenging in studies with laboratory mice. Over the past five decades, several approaches have been developed to maximize the quantity and quality of CSF samples, either from live or euthanized animals. Due to the small amounts collected, samples from single mice were often pooled or diluted to meet volume requirements of automated counters and multiple assays.
NEW METHOD
This paper reviews previous work on CSF collection in mice, thus providing methodological background for the current post-mortem procedure. This modified cisternal puncture method involves the use of a peristaltic pump for consistent and slow intracardiac perfusion, as well as a loupe headset with a custom-made glass pipette for piercing a single hole in the atlanto-occipital membrane during repeated CSF draws. Sample cleanness is verified by comparing the colour of the glass pipette and the bottom of centrifuged PCR vial against a white background.
RESULTS
With three trained experimenters, the entire procedure (including anesthesia) takes ∼11-13 min and often results in the collection of up to 40 μl of clean CSF from males of different murine strains. Properly staggered collections allow processing of relatively large cohorts of mice per day.
CONCLUSIONS
This modification of previously employed methods can be used in studies that require tightly-timed collections of larger volumes of undiluted, tissue-free CSF and/or individual data records.
Topics: Animals; Cerebrospinal Fluid; Cisterna Magna; Mice; Neurosurgical Procedures; Spinal Puncture
PubMed: 30273638
DOI: 10.1016/j.jneumeth.2018.09.025 -
Cold Spring Harbor Protocols Jan 2018Intracytoplasmic sperm injection (ICSI) is the injection of a single spermatozoon directly into the cytoplasm of an oocyte using an injection pipette. Spermatozoa that...
Intracytoplasmic sperm injection (ICSI) is the injection of a single spermatozoon directly into the cytoplasm of an oocyte using an injection pipette. Spermatozoa that are used for ICSI do not need to be motile or alive. Indeed, for mouse ICSI, the sperm tail is typically broken off before injection into the oocyte, resulting essentially in a nuclear transfer. The protocol describes creation of the injection pipettes, preparation of oocytes and spermatozoa, and steps of the ICSI procedure. The technique can be used to create both hybrid and inbred mice, as well as transgenic animals.
Topics: Animals; Female; Male; Mice; Microinjections; Oocytes; Sperm Injections, Intracytoplasmic; Spermatozoa
PubMed: 29295903
DOI: 10.1101/pdb.prot094482 -
Cold Spring Harbor Protocols Aug 2022An essential requirement of every laboratory procedure is to have all materials ready when they are needed, so that the experimental flow is not disrupted. This is...
An essential requirement of every laboratory procedure is to have all materials ready when they are needed, so that the experimental flow is not disrupted. This is particularly true for patch clamping; therefore, effort must be devoted in advance to produce materials such as patch pipettes. This can be a fiddly business; hence, this protocol provides step-by-step advice on how to pull and polish patch-clamp pipettes. It also includes a brief description on how to prepare homemade filling devices to deliver saline efficiently and inexpensively into the pipettes. The protocol ends with guidelines on how to change the filament of a Sutter horizontal puller, a dreaded yet necessary activity that should be learned by anyone who wishes to become an expert patch clamper.
Topics: Animals; Constriction; Drosophila; Neurons; Patch-Clamp Techniques
PubMed: 35798463
DOI: 10.1101/pdb.prot107932