An Air Pouch Mouse Model for Lipopolysaccharide-Induced

Inflammatory Exudate Collection

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Transcript

After anesthetization of the mice on day zero, use a 0.22-micron filter attached to

a five-milliliter syringe to obtain a three-milliliter volume of sterilized air. Lift the

back skin of the anesthetized mouse with tweezers and subcutaneously use a 26-

gauge by 3/8th-inch needle to inject three milliliters of the sterilized air.

After treatment, remove the mice from the breathing unit and place them into a

well-set cage. Monitor the mice to ensure they are alive until they start to move

around. On day three, inject an additional three milliliters of sterilized air into the

previously-established air pocket to sustain the air pouch.

On day six, inject different treatments into the air pouch. Inject one milliliter of PBS

as a negative control, and inject one milliliter of one microgram per milliliter LPS as

the positive control to induce local inflammation. After six hours, sacrifice the mice.

For each air pouch, inject one milliliter of wash buffer to wash the air pouch and

collect the inflammatory exudate in a 15-milliliter centrifuge tube. Then, wash the

air pouch with two milliliters of wash buffer twice, and collect the inflammatory

exudate in the same centrifuge tube.

Centrifuge at 100 x g for 10 minutes at room temperature. Discard the supernatant

and resuspend the cells in one milliliter of wash buffer. Count the cells to quantify
the neutrophil ratio, using the automatic hematology analyzer.