An Air Pouch Mouse Model for Lipopolysaccharide-Induced Inflammatory Exudate Collection

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Air pouch assay

1. First air injection
   1. On day 0, fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min.
      NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.
   2. Use a 0.22 µm filter attached to a 5 mL syringe to obtain a 3 mL volume of sterilized air.
   3. Lift the back skin of the anesthetized mouse with tweezers and subcutaneously inject 3 mL of sterilized air using a 26 G x 3/8" needle.
   4. After treatment, remove the mice from the breathing unit. Monitor the mice to ensure they are alive until they start to move around.
2. Second air injection
   1. On day 3, inject an additional 3 mL of sterilized air into the previously established air pocket to sustain the air pouch as described in section 1.1.
3. Treatment
   1. On day 6, 6 h before sacrifice, inject different treatments into the air pouch. Inject 1 mL of phosphate-buffered saline (PBS) as a negative control. Inject 1 mL of 1 µg/mL LPS as the positive control to induce local inflammation.
   2. Fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min. Prepare wash buffer according to Table of Materials.
      NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.
   3. For each air pouch, wash the air pouch with 1 mL of wash buffer and collect the inflammatory exudate in a 15 mL centrifuge tube. Wash the air pouch with 2 mL of wash buffer 2x and collect the inflammatory exudate in the same centrifuge tube.
   4. Centrifuge at 100 x g for 10 min at RT. Discard the supernatant and resuspend cells in 1 mL of wash buffer. Count the cells to quantify the neutrophil ratio using the automatic hematology analyzer.
      NOTE: See representative results in Figure 1.