["Collaborating Expert | M.S. Ma Xiurong", "Medicinal Chemistry Kunming University of Science and Technology"], ["Reviewed by | Dr. Guo Ya-jie", "Biochemistry Molecular Biology University of Chinese Academy of Sciences"]
Summary
Nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammatory vesicles have been well studied in recent years. NLRP3 is a multi-protein complex, which is involved in the development of various diseases by regulating the activation of caspase-1, inducing the maturation and secretion of inflammatory factors IL-1β and TNF-α, and promoting inflammation and pyroptosis.
Principle
Rapid opening of extracellular adenosine triphosphate (ATP)-gated ions can lead to exocytosis of intracellular K+, which in turn induces activation of NLRP3. Common endotoxins, such as lipopolysaccharide (LPS), can activate the synthesis and release of multiple cytokines and inflammatory mediators intracellularly through cellular signaling, causing a series of responses, and the activation of NLRP3 inflammatory vesicles can be detected by using treatments such as ATP and LPS.
Operation method
Activation and detection of NLRP3 inflammatory vesicles
Principle
Rapid opening of extracellular adenosine triphosphate (ATP)-gated ions can lead to exocytosis of intracellular K+, which in turn induces activation of NLRP3. Common endotoxins, such as lipopolysaccharide (LPS), can activate the synthesis and release of multiple cytokines and inflammatory mediators intracellularly through cellular signaling, causing a series of responses, and the activation of NLRP3 inflammatory vesicles can be detected by using treatments such as ATP and LPS.
Materials and Instruments
Cell culture incubator, enzyme marker, laser confocal microscope, mouse macrophages (RAW264.7), LPS, ATP, DMEM medium, fetal bovine serum, penicillin, streptomycin, 0.25% trypticase-EDTA solution, Elisa assay kits for IL-1β and TNF-α, and propidium iodide (PI) staining.
Move
(1) Cell treatment: RAW264.7 was inoculated in 6-well plates and cultured to 85%, then the cells were randomly divided into the control group, the LPS-treated group and the LPS + ATP co-treated group. In the control group, the cells were washed once with PBS, replaced with culture medium, and continued to be cultured for 6 h. In the LPS-treated group, the cells were washed once with PBS, replaced with culture medium containing 500 ng/mL LPS for 6 h. In the co-treated group, the cells were washed once with PBS, replaced with culture medium containing 500 ng/mL LPS, and continued to be stimulated for 30 min with the addition of 2 mM ATP without replacing the culture medium for 5.5 h. The treatment was completed with the addition of 2 mM ATP. After the treatment, the cell supernatant was collected for ELISA.
(2) ELISA for IL-1β and TNF-α: The content of inflammatory factors IL-1β and TNF-α was detected strictly according to the instructions of Elisa kit, and the antibody was encapsulated in a flat-bottomed, half-zone, transparent 96-well plate, and the absorbance of the samples was detected at 450 nm using an enzyme counter.
(3) Detection of cell death by PI staining: The cell treatment method was the same as that in step 1. After treatment, 40 μg/mL of PI stain was prepared to treat the cells, and the cells were incubated at room temperature and protected from light for 20 min, then the stain was removed and washed twice with PBS, and the cell death was observed under a confocal laser microscope.
(4) Data processing: The data obtained were analyzed by SPSS18.0 software. Step 2: The model was established on the basis that the control group did not contain IL-1β and TNF-α; the LPS-treated group did not contain IL-1β, but the content of TNF-α increased with the stimulation of LPS, which was more obvious than that of the control group; in the group co-treated with LPS + ATP, the content of both IL-1β and TNF-α increased, which was more obvious than that of the control group. Step 3 was established on the basis of the red coloration of dead cells detected by laser confocal microscopy under 535 nm light excitation.
Caveat
1、Wash the cells with PBS and add the staining agent gently to avoid the cells blowing up and affecting the experimental results.
2、PI staining solution should be used now and avoid light to prevent fluorescence quenching.
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