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MindMap Gallery Establishment and identification of endotoxic shock mouse model

Establishment and identification of endotoxic shock mouse model

This is a mind map about the establishment and identification of endotoxin shock mouse model. It is introduced in detail and comprehensively described. I hope it can be helpful to interested friends.

Edited at 2023-11-29 21:19:05

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Establishment and identification of endotoxic shock mouse model

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Establishment and identification of endotoxic shock mouse model

Establishment of LPS-induced endotoxic shock mouse model

septicemia

Refers to an acute systemic infection caused by pathogenic bacteria invading the blood circulation and growing and multiplying.

In sepsis caused by Gram-negative bacteria, endotoxin is the most critical activating factor

LPS is the main component of endotoxin and can cause inflammatory reactions

After gram-negative bacteria infect the body, they release a large amount of endotoxin, which acts on the body's macrophages, inducing cell activation and producing biologically active substances.

Active substances act on microvessels and cause functional disorders, leading to microcirculation disorders.

Clinical manifestations include microcirculation failure, hypotension, hypoxia, acidosis, etc. Severe cases can lead to shock.

Intraperitoneal injection of LPS is used to simulate the process of shock caused by Gram-negative bacterial infection and the release of large amounts of endotoxin in the body.

LPS can be used as a late model of septic shock 6 hours after intraperitoneal injection

Isolation and in vitro stimulation of mouse peritoneal macrophages

CD14 and TLR4 expressed on the surface of macrophages are very important receptors for LPS

After LPS is transported by CD14, it combines with TLR4 and activates the intracellular signaling pathway, causing macrophages to change from a resting state to an activated state. Their phagocytosis, killing, antigen-presenting and immune-regulating functions are all enhanced. .

After intraperitoneal injection of LPS, the macrophages resident in the peritoneal cavity are activated by LPS, become activated macrophages, and begin to secrete a variety of cytokines and chemokines.

These chemokines and cytokines can further chemoattract macrophages in the peripheral blood into the peritoneal cavity and activate them.

A large number of activated macrophages accumulate in the peritoneal cavity

After peritoneal macrophages are isolated and re-stimulated with LPS in vitro, high levels of intracellularly stored IL-1β and other cytokines will be released within a short period of time.

Extraction and reverse transcription of total RNA from mouse peritoneal macrophages

Extraction of total RNA

Since RNase A enzymes rely on histidine residues in the active site for catalysis, they can be inhibited by the histidine alkylating agent diethyl pyrocarbonate.

Total RNA isolated from eukaryotic cells mainly contains non-coding RNA, such as ribosomal RNA and transfer RNA, as well as messenger RNA

mRNA is the template for protein biosynthesis and its abundance is low, accounting for only 2% to 5% of the total weight of cellular RNA.

Extraction of total RNA from animal cells using TRIzol method

The main ingredients in TRIzol reagent are guanidine isothiocyanate and phenol

Guanidine isothiocyanate can lyse cells, promote the cleavage of ribosomes, separate RNA from proteins, and release RNA into the solution. At the same time, it effectively inhibits the activity of RNase, thereby maintaining the integrity of RNA.

Chloroform can extract acidic phenol, and acidic phenol can cause RNA to enter the aqueous phase. After centrifugation, the homogenate can be divided into a transparent upper aqueous phase layer (containing RNA), a phase interface, and a red lower organic layer (containing DNA and protein).

Isopropyl alcohol precipitates RNA from the aqueous layer

Reverse Transcription

Using mRNA as a template, first reverse-transcribe to generate cDNA, and then perform PCR amplification on the cDNA.

Reverse transcriptase has three activities: RNA-guided DNA polymerase activity, DNA-guided DNA polymerase activity, and RNase H activity

Using RNA as a template and dNTP as a substrate, under the catalysis of reverse transcriptase and the guidance of primers, a DNA single strand complementary to the RNA template is synthesized, which forms an RNA/DNA hybrid double strand with the RNA template.

Under the action of reverse transcriptase, the RNA strand is hydrolyzed, and the remaining single-stranded DNA is used as a template to synthesize the second strand. The double-stranded DNA synthesized for the second time is cDNA.

PCR amplification of peritoneal macrophage IL-1B gene

PCR

transsexual

After the template DNA is heated to about 94°C for a certain period of time, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated and turned into a single strand so that it can bind to the primer.

annealing

After the template DNA is heated and denatured into a single strand, the temperature drops to about 55°C, and the primer pairs with the complementary sequence of the template DNA single strand.

extend

Under the action of Taq enzyme, the DNA template-primer conjugate uses dNTP as the reaction raw material and the target sequence as the template. According to the principles of base pairing and semi-conservative replication, a new semi-conservative replication strand complementary to the template DNA strand is synthesized.

DNA agarose gel electrophoresis

Many agarose chains coil around each other under the action of hydrogen bonds and other forces to form rope-like agarose bundles, forming a large mesh gel.

DNA is negatively charged under alkaline conditions (pH 8.0 buffer) and moves toward the positive electrode through the gel medium in the electric field. Different DNA molecule fragments have different swimming rates in the electric field due to different molecules and configurations.

The fluorescent dye generates fluorescence under the excitation of ultraviolet light. When the DNA sample swims from the negative electrode to the positive electrode in the agarose gel, the fluorescent dye moves from the positive electrode to the negative electrode.

ELISA to detect IL-1B content in macrophage culture supernatant

Use purified rabbit anti-mouse IL-1β antibody to coat the microwell plate to make a solid-phase antibody. Add mouse macrophage culture supernatant to the microwells coated with monoclonal antibody.

Add biotin-labeled rabbit anti-mouse IL-1β antibody to form an antibody-antigen-biotin antibody complex. After washing, add avidin-horseradish peroxidase complex to wash away the excess label. At this time, the solid The amount of enzyme in the phase is directly proportional to the amount of the substance being tested in the specimen.

The enzyme reaction substrate ABTS is added, and the enzyme catalyzes the substrate to form a colored product. The color of the product can be quantitatively analyzed with a microplate reader to determine the amount of IL-1β in the sample.