Encyclopedia of Experiments: Immunology
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Transcript
Place an anesthetized mouse on the sterile surgical pad attached to a breathing unit with a continuous flow of oxygen and an anesthetic gas.
Using a filter, load the syringe with sterile air. Attach a needle to the syringe and inject sterilized air subcutaneously into the back of the mouse to create an air pouch within the tissue layers. Monitor the mouse until recovery.
Re-anesthetize the mouse and administer lipopolysaccharide, LPS, into the air pouch. LPS binds to the toll-like receptor 4, TLR-4 on the tissue-resident macrophages and dendritic cells, activating them.
The activated cells release various inflammatory mediators like cytokines and chemokines, that attract neutrophils and other immune cells. These mediators also increase vascular permeability of the nearby blood vessels, enabling immune cells and soluble factors to enter the air pouch from the bloodstream.
As fluid accumulates within the pouch, it forms an exudate comprising inflammatory cells and various bioactive molecules.
Euthanize the mouse and inject a suitable wash buffer into the air pouch. Collect the inflammatory exudate into a microcentrifuge tube and centrifuge. Discard the supernatant and resuspend the cells in the wash buffer.
Analyze the inflammatory exudate to quantify the neutrophil ratio and determine the extent of their infiltration.
