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Protocol
Primary Intestinal Fibroblasts: Isolation, Cultivation, and Maintenance
Intestinal fibroblasts maintain homeostasis and contribute to inflammatory responses and the development of cancer. Intestinal fibroblasts express pattern recognition receptors which can mount an immune respon...
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Protocol
Biogenesis of Mesoporous Silica Nanoparticles Enclosed in Extracellular Vesicles by Mouse Renal Adenocarcinoma Cells
Integrating the versatility of synthetic nanoparticles to natural biomaterials, such as cells or cell membranes, has gained considerable attention as promising alternative cargo delivery platforms in recent ye...
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Protocol
TaqMan Real-Time RT-PCR Detection of Porcine Epidemic Diarrhea Virus
Porcine epidemic diarrhea virus (PEDV) is a highly infectious agent that causes the fatal enteric disease porcine epidemic diarrhea (PED) in neonatal pigs. Rapid detection and quantitation of PEDV are critical...
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Protocol
RPLC-RPLC-MS/MS for Proteoform Identification
Top-down proteomics methods have a distinct advantage over bottom-up methods in that they analyze intact proteins rather than digested peptides which can result in loss of information regarding the intact prot...
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Protocol
Affinity-Based Enrichment of Extracellular Vesicles with Lipid Nanoprobes
Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles with sub-micrometer size that are released by various cells. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic a...
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Protocol
Electrochemical Assay for Continuous Monitoring of Dynamic DNA Methylation Process
DNA methylation is an important mode of epigenetic modification, which plays key roles in many cellular processes. Within the last decades, various methods have been proposed for quantitative analysis of methy...
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Protocol
Design of an Intron-Retained Bioluminescence Reporter and its Application in Imaging of Pre-mRNA Splicing in Living Subjects
Aberrant splicing of precursor messenger RNA (pre-mRNA) can generate abnormal transcripts, and most of the human diseases have been shown to associate with abnormal splicing of pre-mRNA. Conventional methods r...
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Protocol
Accurate Proteoform Identification and Quantitation Using pTop 2.0
The remarkable advancement of top-down proteomics in the past decade is driven by the technological development in separation, mass spectrometry (MS) instrumentation, novel fragmentation, and bioinformatics. H...
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Protocol
CRISPR-Based Transcriptional Activation in Drosophila
Overexpression is one of the classical approaches to study pleiotropic functions of genes of interest. To achieve overexpression, we often increase the transcription by introducing genes on exogenous vectors o...
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Protocol
CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440
Genome editing technologies allow us to study the metabolic pathways of cells and the contribution of each associated enzyme to various processes, including polyhydroxyalkanoate (PHA) synthesis. These biodegra...
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Protocol
RNA-Sequencing and Transcriptome Analysis after Fluid Shear Stress Stimulation in Lymphatic Endothelial Cells
Fluid shear stress (FSS) is an important mechanical force that regulates endothelial and vascular functions in the blood and lymphatic vasculature. RNA-sequencing (RNA-Seq), an emerging next-generation sequenc...
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Protocol
In Vitro Study of Permeability in Lymphatic Endothelial Cells Responding to Histamine
Histamine is well characterized to cause hyperpermeability in both blood and lymphatic endothelial cells (BECs and LECs) in infection and inflammation. The increased permeability impairs the barrier function o...
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Protocol
Metabolomic Analysis of Natural Variation in Arabidopsis
Methodological advances in coupled-mass spectrometry (gas chromatography and liquid chromatography; GC-MS and LC-MS) have rendered the profiling of highly complex plant extracts relatively facile and allowed t...
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Protocol
FREMSA: A Method That Provides Direct Evidence of the Interaction between microRNA and mRNA
microRNAs (miRNAs) modulate the expression of enzymes responsible for activation or detoxification of xenobiotics and toxicants. miRNAs are dysregulated in response to environmental exposure and have been impl...
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Protocol
Methods for Establishing and Using a Stable Cell Line Expressing Both Gaussia Luciferase and Firefly Luciferase to Screen for Endoplasmic Reticulum Stress
Endoplasmic reticulum (ER) stress is one of the major mechanisms underlying the etiology of multiple diseases and drug-induced toxicity. Gaussia luciferase (Gluc) is a naturally secreted protein that has been use...
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Protocol
Using a Lentivirus-Based Inducible RNAi Vector to Silence a Gene
RNA interference (RNAi) is a powerful approach for inhibiting gene expression and its wide applications have expanded our understanding of gene functions. Short hairpin RNAs (shRNAs) are artificially synthesiz...
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Protocol
Development of Pico-ESI-MS for Single-Cell Metabolomics Analysis
In this chapter, we introduced a Pico-ESI strategy for metabolomics analysis with picoliter-level samples. This Pico-ESI strategy was technically achieved by pulsed direct current electrospray ionization sourc...
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Protocol
Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9
Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms inc...
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Protocol
Application of Whole-Genome Sequencing to Transposon Mutants of Salmonella Heidelberg
Transposons are elements widely dispersed among organisms which are able to move and replicate fragments of genomes. The extensive variability in transposons present in most organisms requires extensive identi...
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Protocol
CRISPR/Cas9 for Mutagenesis in Rice
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has be...
