Methods in molecular biology
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Despite advances in intensive care unit interventions, including the use of specific antibiotics and anti-inflammation treatment, sepsis with concomitant multiple organ failure is the most common cause of death in many acute care units. In order to understand the mechanisms of clinical sepsis and develop effective therapeutic modalities, there is a need to use effective experimental models that faithfully replicate what occurs in patients with sepsis. Several models are commonly used to study sepsis, including intravenous endotoxin challenge, injection of live organisms into the peritoneal cavity, establishing abscesses in the extremities, and the induction of experimental polymicrobial peritonitis via cecal ligation and puncture (CLP). Here, we describe the surgical procedure of CLP in mice, which has been demonstrated to closely replicate the nature and course of clinical sepsis in human subjects.
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Neutrophils are some of the first leukocytes to respond to inflammatory stimuli. Once recruited, these cells are equipped with an assortment of proteolytic enzymes and antimicrobial factors that disarm and degrade pathogens. ⋯ In this chapter, we describe protocols to isolate bone marrow-derived neutrophils from mice. We further describe in vitro methods to spectrophotometrically quantify, immunolabel, and visualize NET structures.
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Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. ⋯ We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.
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DNA methylation changes are dynamic processes which occur at cytosines of CpG dinucleotides and contribute to normal development but also to diseases. DNA methylation changes are most effective in promoters and enhancers, the former frequently being CpG-rich and the latter, in contrast, CpG-poor. Many genome-wide methods for DNA methylation analysis interrogate predominantly CpG-rich regions and, hence, spare enhancers and other potentially important genomic regions. ⋯ In a subsequent step, the non-covalently bound adapter oligonucleotide needs to be replaced by a novel oligonucleotide to provide the proper adapter sequence for the reverse strand in paired-end sequencing. The presented protocol describes an improved, simplified version of TWGBS where the inefficient oligo-replacement is circumvented by usage of a sequencing-compatible transposase-adapter complex. Consequently, genomic DNA of only a few hundred human cells is required to interrogate the complete human DNA methylome.
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The protocol presented was specifically optimized for in-depth analysis of the human colon mucosa proteome. After cell lysis in a sodium deoxycholate/urea buffer, a tandem digestion with Lys-C and trypsin was performed. Prior to LC-MS/MS analysis, peptides were TMT-labeled and fractionated by high pH reversed-phase spin columns. This protocol is a powerful, reproducible, sample-saving, and cost-effective option when an in-depth quantitative proteome analysis is desired.