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.
