Chinese Academy of Sciences

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Ca²⁺ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca²⁺ signals in Arabidopsis cells and tissues using the genetically encoded Ca²⁺ indicators Aequorin or Case12.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

We present a protocol on modular design and production of intelligent robots to help scientific and technical workers design intelligent robots with special production tasks based on personal needs and individualized design.

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Dormant and active cancer cell phenotypes were characterized using quantitative phase imaging. Cell proliferation, migration, and morphology assays were integrated and analyzed in one simple method.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified λ DNA substrate and a Quantum-dot labeled protein.

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1^-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

A protocol to create a full-range linear displacement sensor, combining two packaged fiber Bragg grating detectors with a magnetic scale, is presented.

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Precise determination of the evolved gases' flow rate is key to study the details of reactions. We provide a novel quantitative analysis method of equivalent characteristic spectrum analysis for thermogravimetry-mass spectrum analysis by establishing the calibration system of the characteristic spectrum and relative sensitivity, for obtaining the flow rate.

Diffusive convection (DC) widely occurs in natural processes and engineering applications, characterized by a series of staircases with homogeneous convecting layers and stratified interfaces. An experimental procedure is described to simulate the evolution process of the DC staircase structure, including the generation, development and disappearance, in a rectangular tank.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Oxide materials show many exotic properties that can be controlled by tuning the oxygen content. Here, we demonstrate the tuning of oxygen content in oxides by varying the pulsed laser deposition parameters and by performing postannealing. As an example, electronic properties of SrTiO₃-based heterostructures are tuned by growth modifications and annealing.

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

The protocol describes a method to purify and separate the U and Th nuclide in submarine hydrothermal sulfide sample with Fe co-precipitation and extraction chromatography for ²³⁰Th-U disequilibrium dating.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

A protocol for the space payload design, the space experiment on thermocapillary convection, and analyses of experimental data and images are presented in this paper.

Many researchers generate "medium-sized", low-velocity, and multi-dimensional data, which can be managed more efficiently with databases rather than spreadsheets. Here we provide a conceptual overview of databases including visualizing multi-dimensional data, linking tables in relational database structures, mapping semi-automated data pipelines, and using the database to elucidate data meaning.

A protocol that uses enhanced QM/MM method to investigate the isotopic effect on the double proton transfer process in porphycene is presented here.

Herein, a protocol to conduct the Morris water maze tests to evaluate the ability of learning and memory of Alzheimer’s Disease model mice and to assess the effect of manual acupuncture for treating them is described.

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre^+/-;Rosa26^RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

This protocol describes a method to determine the influence of ryegrass residue addition on soil organic matter mineralization (i.e., priming effect) as well as explore the changes in soil microbial biomass size induced by soil organic matter priming, which involves artificially changing the size of microbial biomass.

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential β₂-microglobulin (β₂m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous β₂m were investigated.

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

A technique utilizing a solid fuel grain with a novel nested helical structure to improve the combustion performance of a hybrid rocket engine is presented.

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

The presented protocol integrates various evaluation methods and demonstrates a method to evaluate the keyboard design on smartphones. Pairs matched by English characters are proposed as the input material, and the transition time between two keys is used as the dependent variable.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Traceability calibration of mechanical characteristics of thrust stand is an essential prerequisite to ensure traceability measurement of thrust. Here, we describe how to calibrate the thrust stand by the electrostatic force generated by the parallel plate capacitor.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 µm tissue sections under confocal microscopy.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

This protocol presents the clinical application of a 24 G cannula and 3-0 polypropylene suture as a simple and effective method for the exploration of the vas deferens.

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

The secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol describes how to assess the impact of xenobiotics on alfalfa via nontargeted metabolomic analysis.

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

Nasonia wasp embryos were dissected from Lucillia sericata pupae after parasitization for 12-24 h and washed with alcohol and 10% sodium hypochlorite solution to obtain germ-free embryos. After rearing the germ-free embryos and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

Here, we introduce three methods for the physical control of pest rodents, four methods for counting their effectiveness against rodents, and the statistics of the effect of building a rodent-proof wall.

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

Exosomes possess significant clinical potential, but their practical application is limited due to easy in vivo clearance and poor stability. Microneedles present a solution by enabling localized delivery by puncturing physiological barriers and dry-state preservation, thereby addressing the limitations of exosome administration and expanding their clinical utility.