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Protocol
Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry Analysis Directly in Gas Exchange Cuvette
Isotopically nonstationary metabolic flux analysis (INST-MFA) is a powerful technique for studying plant central metabolism, which involves introducing a 13CO2 tracer to plant leaves and sampling the labeled meta...
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Protocol
Using Dynamic Gas Exchange Measurements During Oxygen Transients to Study Nonsteady-State Photorespiration
Leaf-level gas exchange is widely used to investigate the largest carbon fluxes in illuminated leaves, offering a nondestructive way to investigate the impact of photorespiration on plant carbon balance. Moder...
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Protocol
Hyperspectral Proximal Sensing for Estimating Photosynthetic Capacities at Leaf and Canopy Scales
Agronomists, plant breeders, and plant biologists have been promoting the need to develop high-throughput methods to measure plant traits of interest for decades. Measuring these plant traits or phenotypes is ...
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Protocol
Antibody Display Technology (ADbody) to Present Challenging and Unstable Target Proteins on Antibodies
Antibodies are the major components of adaptive immunity for the recognition of diverse antigens. Six complementarity-determining regions (CDRs) from each heavy chain and light chain present the antigen-bindin...
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Protocol
Investigation of ROP GTPase Activity and Cytoskeleton Dynamics During Tip Growth in Root Hairs and Pollen Tubes
Pollen tubes and root hairs are typical tip-growing cells and are employed as model systems to study plant cell polarity. Previous studies have shown that the Rho family ROP GTPase plays a critical role in the...
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Protocol
Single-Cell Quantification of the Mechanical Stability of Cell–Cell Adherens Junction Using Glass Micropipettes
Micropipette-based methods have been widely used for the manipulation of cells and characterization of the mechanical properties at the cell or tissue level. Here, we introduce the glass micropipette-based mec...
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Protocol
Adoptive Transfer of Cryptococcus neoformans-Specific CD4 T-Cells to Study Anti-fungal Lymphocyte Responses In Vivo
CD4 T-cells are important for long-term control and clearance of several fungal infections in humans, particularly those caused by Cryptococcus species. Understanding the mechanisms underlying protective T-cell i...
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Protocol
Imaging of Cortical Microtubules in Plants Under Salt Stress
The microtubule (MT) network is a highly dynamic subcellular structure playing an important role in the growth and development of plants, and it is able to respond to biotic and abiotic environmental signals. ...
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Protocol
Dense and Sparse Labelling of Mitral Cells by Oral and Intraperitoneal Routes of Tamoxifen Administration
Cell type-specific labelling and manipulation using Cre-driver lines have become integral to analyses of neuronal circuits in the brain. To study how mitral cells of the olfactory bulb process olfactory inform...
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Protocol
Multiple Electrode Aggregometry (Multiplate): Functional Assay for Vaccine-Induced (Immune) Thrombotic Thrombocytopenia (VITT)
Vaccine-induced immune thrombotic thrombocytopenia (VITT) was first described in 2021 and represents an adverse reaction to adenoviral vector COVID-19 vaccines AstraZeneca ChAdOx1 nCoV-19 (AZD1222) and Johnson...
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Protocol
Synthetic Biology Meets Machine Learning
This chapter outlines the myriad applications of machine learning (ML) in synthetic biology, specifically in engineering cell and protein activity, and metabolic pathways. Though by no means comprehensive, the...
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Protocol
In-Depth Blood Proteome Profiling by Extensive Fractionation and Multiplexed Quantitative Mass Spectrometry
Blood in the circulatory system carries information of physiological and pathological status of the human body, so blood proteins are often used as biomarkers for diagnosis, prognosis, and therapy. Human blood...
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Protocol
Isolation and Mass Spectrometry-Based Profiling of Major Lipids in Brown Adipose Tissue
Brown adipose tissue (BAT) is an important regulator of metabolic homeostasis through its role in adaptive thermogenesis and control of whole-body glucose metabolism. Lipids play multiple roles in BAT function...
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Protocol
Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration
Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with...
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Protocol
Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles
Galectins are animal lectins that recognize β-galactoside and bind glycans. Recent studies have indicated that cytosolic galectins recognize cytosolically exposed glycans and accumulate around endocytic vesicl...
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Protocol
Quantification of In Vitro Blood-Brain Barrier Permeability
In vitro blood-brain barrier (BBB) models have been widely used to simulate in vivo models due to their low cost, feasibility, and repeatability. To serve as a valid substitute, the in vitro BBB should have th...
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Protocol
Automated Synthesis of Chondroitin Sulfate Oligosaccharides
Glycosaminoglycans (GAGs) are important sulfated carbohydrates prevalently found in the extracellular matrix that serve many biological functions. The synthesis of structurally diverse but defined GAGs is extr...
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Protocol
Collagen Sandwich Culture of Primary Hepatocytes for Image-Based Investigations
Collagen sandwich culture of hepatocytes retains many in vivo-like properties and is used for many investigations in liver cell biology, and hepatic pharmacology and toxicology. This chapter describes the meth...
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Protocol
Map** R-Loops Using Catalytically Inactive RNaseH1 (R-ChIP)
R-loops, three-stranded structures containing double-stranded DNA invaded by single-stranded RNA, have been linked to diverse biological processes. They play important roles in regulating gene regulation and D...
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Protocol
One-Pot Enzymatic Synthesis of Heparin from N-Sulfoheparosan
Heparin, a glycosaminoglycan-based anticoagulant drug, is prepared as an extract of animal tissues. Heparosan, an Escherichia coli (E. coli) K5 capsular polysaccharide with the structure →4)-β-D-glucuronic acid (...
