Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

The overall aim of this procedure is to non-surgically still test material into the lungs of mice using a gavage needle and to subsequently analyze the lungs and lung draining lymph nodes. This is accomplished by first inserting an illuminating laryngoscope and bent gavage needle into the trachea of an anesthetized mouse. The test material is then instilled into the lungs of the animal via the laryngoscope and gavage needle.

Next, the lungs and draining lymph nodes are extracted. Finally, the lungs and lymph nodes are processed into single cell suspensions. Ultimately, this method facilitates flow cytometric analysis of the association of the test material with lung immune cells and of the trafficking of the cells to the lung draining lymph nodes.

The main advantage of this technique over existing methods like intranasal and aerosolized exposures, is that it allows directed delivery of the test material into the lungs bypassing the nasal associated lymphoid tissue. In addition, it's simple to perform compared to surgical rats of in tracheal installation. Demonstrating the procedure will be Elizabeth Enti, a postdoc in the Richie's lab, and Manji a graduate student in the lens lab.

After anesthetizing the mouse, prepare the inoculum in 50 microliters of saline. Fill a one milliliter syringe fitted with a sterile bent gavage needle with the inoculum. Load the syringe with a 100 microliter air pocket behind the inoculum to ensure that all of the fluid is installed into the lung.

Place the mouse on the angled wooden platform hanging by its incisors on the wire and gently restrain it in place with a piece of ribbon. Next turn on the laryngoscope with one hand and use the other hand to grab a pair of blunt tended forceps. Use the tip of the laryngoscope and the forceps to gently pry open the mouth with the forceps.

Pull the tongue out and hold it to the side. Guide the laryngoscope blade towards the back of the mouth. Keep the laryngoscope pressed down very gently at a 90 degree angle until the opening of the trachea can be seen.

While holding the laryngoscope in place with one hand, take the one milliliter syringe fitted with the bent gavage needle containing the inoculum with the other hand. Insert the needle into the trachea until the bend in the needle is by the front incisors. Push the plunger evenly to deliver the inoculum without bubbling.

Pull the needle outta the trachea as soon as possible. Hold the mouse upright for a few seconds to allow the inoculum to be inhaled into the lungs. Place the mouse near a heating lamp and allow it to wake up.

After euthanizing pin the arms and legs of the mouse to a dissection board. Make an incision along the middle of the ventral side with scissors. Gently pull the skin and expose the peritoneum.

Make incisions on the peritoneum to expose the abdominal organs. Hold the tip of the sternum with the forceps and puncture the diaphragm with the scissors. Cut the diaphragm along the sides of the rib cage with the scissors parallel to the dissection board surface.

Cut through the thoracic cavity along the dorsa ventral line on both sides slightly. Lift up the ribcage on the right side of the heart. Just underneath the thymus look for two lymph nodes.

A third slightly larger lymph node is located underneath a small arterial blood vessel that runs perpendicular to the trachea. Harvest the lymph nodes into one milliliter of lymph node digestion, mix and place on ice. After harvesting the lymph nodes, harvest the lungs into a six well plate.

Keeping the six well plate on ice aspirate the PBS and chop the lobes into small pieces using forceps and scissors. Hold each lymph node down with a 26 gauge needle and tease it apart with a second 26 gauge needle. To ensure the release of all cells, add three milliliters of the digestion mix for lungs and one milliliter for the lymph nodes.

After incubating the lymph nodes for 25 to 30 minutes, and the lungs for 30 minutes at 37 degrees Celsius, add EDTA to a final concentration of 10 millimolar to each of the digestion mixtures to stop the reaction and then place them on ice. Using a three milliliter syringe, pass pieces of the lungs through an 18 gauge needle. Three times mash the pieces of the lungs and the lymph nodes through 70 to 100 micron cell strainers.

Using the back end of a one milliliter syringe plunger, wash the cell strainers with 10 milliliters of HBSS without calcium and magnesium centrifuge. The cell samples at 500 times gravity for five minutes at four degrees Celsius. Reese's bend the pellets in five milliliters of red blood cell lysis, buffer, and incubate for three minutes.

At room temperature, add 10 milliliters of HBSS without calcium and magnesium to quench the lysis centrifuge the samples at 500 times gravity for five minutes. Bring up the lung and the lung draining lymph nodes, single cell suspensions to 10 milliliters and five milliliters respectively. Use triam blue exclusion in a hemo cytometer to count cells.

In these two figures, AJ mice were challenged with one times 10 to the eighth basillas rassis spores, and the lungs were analyzed by flow cytometry at 30 minutes post-infection. These dot plots show the gating strategy. These panels show the percentage of CD 45 positive CD 11 C positive lung cells that are positive for bacillus anthrax spores.

In this figure, AJ mice were injected intratracheally and then their lung draining lymph nodes were analyzed. Representative dot plots show the percentage of CD 11 C high cells in the lung draining lymph nodes that harbor fluorescent beads plotted against an empty channel. The left panel shows results from mice that were injected with PBS, and the right panel shows results from mice that were injected with five times 10 to the nine floras, bright yellow green microspheres with 10 micrograms of LPS.

This method can help answer key questions in the field, such as what events are specifically initiated in the lungs, and how inhaled antigens are trafficked into the draining lymph nodes from the lungs.