Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Baylor Research Institute.

1. Culturing Non-typeable Haemophilus influenzae (NTHi) and Preparing the Inoculum

1. Plate NTHi on a chocolate agar plate and keep the plate upside down in a humidified CO₂ incubator overnight at 37 °C and 5% CO₂. The following day, culture the bacteria in brain heart infusion broth. Then, add 1 ml of the broth into a sterile 1.5 ml tube.
2. Centrifuge at 4,000 x g for 5 min at room temperature. Then, discard the supernatant and re-suspend the pellet in 1 ml of sterile 1x PBS. Repeat this step once.
3. Transfer 100 µl of re-suspended solution into 900 µl of 1x PBS and measure the absorbance of the diluted culture at 600 nm wavelength in a spectrophotometer.
4. Determine the viability and colony forming units (CFUs) of the inoculum by culturing 1:10 serial dilutions on chocolate agar plates and incubating overnight at 37 °C and 5% CO₂.
5. Based on the viability and CFUs determined from the previous step, adjust the inoculum to a final concentration of 20 x 10⁶ CFU/ml.

2. Intra-tracheal (i.t) Instillation

1. Inject the mouse intraperitoneally (i.p) with 0.1 ml/10 g body weight of mouse with a 150 mg/ml ketamine + 20 mg/ml xylazine solution. The procedure should be done in the hood with a light source to keep the animal warm.
2. Pinch the interdigital space of the mouse to verify that the mouse is adequately anesthetized.
3. Place the mouse on its back. Shave the ventral area of the neck and disinfect with chlorhexidine and 70% ethyl alcohol.
4. Lift the skin of the upper ventral part of the neck using rat tooth forceps. Make a small incision (0.5-1.0 cm) above the thymus using a scalpel and carefully dissect the muscle to expose the trachea.
   1. Using a 1 ml syringe with a 27 G needle, slowly inject 0.05 ml air, followed by 0.05 ml NTHi obtained from Section 1 or saline as a control, followed by another 0.05 ml air. Keep animal vertical for around 1 min.
5. Close the incision with surgical glue. Keep the mouse laterally recumbent and warm for about 10 min.

3. BALF Collection

1. Sacrifice the mouse by cervical dislocation 24 hr after infection.
2. Open the abdominal cavity of the mouse with anatomical scissors and cut the ventral aorta to bleed. Expose the ventral part of the lung.
3. Expose the trachea and intubate it with a 20 gauge catheter. Instill 0.8 ml of BALF buffer (1x PBS, 0.1% dextrose, 10 U/ml heparin) and collect the lavage by gentle aspiration. Repeat this procedure three times.
4. Count the total number of cells in 100 µl of BALF suspension using a hemocytometer under 10X magnification with Trypan Blue staining.
5. Prepare slides for modified Giemsa staining. Aliquot 100 µl of BALF into the appropriate wells of the cytospin and centrifuge at 500 x g for 5 min. Dry the slides for 10 min and stain the cells using modified Giemsa stain according to the manufacturer's protocols.
6. Use the remaining cells for subsequent analysis, such as florescence activated cell sorting (FACS), confocal microscopy, gene expression and western blot^11-13.

4. Histopathology Preparation

1. For histopathology analysis, infect mice with NTHi as described in step 2.4. After 24 hr of infection, sacrifice the animals by cervical dislocation. Cut open the thoracic cavity with anatomical scissors to expose the lungs and the trachea as mentioned before. Then, insert the 20 gauge catheter into the trachea.
2. Fill the lung with 2% warm agarose solution prepared in 4% formalin. Once the lungs are fully inflated, place ice on top of the lungs to solidify the agarose solution.
3. Then, carefully remove the thoracic plug and put it in a 50 ml solution of 4% formalin in 1x PBS until it is processed for hematoxylin and eosin (H&E) sections.

5. FACS Staining Cells in the BALF

1. Put 1 x 10⁵ cells from the BALF fluid in a 96-well V-bottom micro titer plate.
2. Wash cells with 300 µl of cold 1x PBS and spin at 4,000 x g for 3 min at 4 °C.
3. Stain the cells with cell stain solution in 100 µl 1x PBS (1:1,000 dilution) and keep at room temperature for 15 min.
4. Wash cells with 300 µl of cold 1x PBS at 4,000 x g for 3 min at 4 °C.
5. Keep cells in 100 µl of FACS wash buffer [Wash buffer (1x PBS, 2% FBS, 1 mM EDTA, 0.1% sodium azide] containing 1:100 diluted Fc-Block for 15 min at 4 °C.
6. Centrifuge cells at 4,000 x g for 3 min and discard supernatant.
7. Stain cells with 100 µl surface staining cocktail (1:100 dilution) in FACS wash buffer for 30 min at 4°C.
8. Wash cells thrice with 300 µl of FACS wash buffer at 4,000 x g for 3 min at 4 °C.
9. Fix cells in 100 µl of 4% paraformaldehyde for 15 min at room temperature.
10. Wash cells twice with 300 µl 1x PBS at 400 x g for 3 min at 4 °C and re-suspend in 300 µl of FACS wash buffer.
11. Make single-color staining controls using flow cytometry beads.
    1. Add one drop of beads into the 96-well V-bottom microliter plate and wash with 200 µl 1x PBS.
    2. Add 1 µl of staining antibody to 100 µl 1x PBS and beads and incubate at room temp for 5 min.
    3. Wash beads twice with 300 µl 1x PBS.
12. Analyze single-stained controls and cells by flow cytometry¹⁴.

6. Homogenization of Lung Tissue to Isolate RNA

1. After infection, collect a small piece of lung (20-40 mg) and keep it in RNA stabilizing reagent at 4 °C overnight. Then, store it at -80 °C until the next step.
2. Wash the lung tissues in cold 1x PBS to get rid of the RNA stabilization reagent.
3. Place the tissue into a 1.5 ml centrifuge tube containing lysis buffer (1 ml lysis buffer + 10 µl β-mercaptoethanol).
4. Chop the tissue into small pieces using pointed scissors and homogenize using a cordless motor pellet pestle for 20-40 sec.
5. Keep the lysate on ice for 5 min and proceed with step 7.1.

7. RNA Isolation from the Lung

1. Centrifuge the lysate at 18,000 x g for 3 min. Remove the supernatant by pipetting and carefully transfer it to a new 1.5 ml tube.
2. Add 1 volume of 70% ethanol to the lysate. Mix immediately by pipetting and wait for 2-4 min.
3. Transfer up to 700 µl of lysate to a spin column placed in a 2 ml collection tube.
4. Centrifuge at 9,600 x g for 15 sec and discard the flow-through.
5. Add 350 µl stringent wash buffer and centrifuge at 9,600 x g for 15 sec. Discard the flow-through.
6. Add 80 µl DNase I directly to the spin column membrane and keep it at room temperature for 15 min.
7. Add 350 µl stringent wash buffer to the spin column and centrifuge at 9,600 x g for 15 sec. Discard the flow-through.
8. Add 500 µl RNA wash buffer and centrifuge at 9,600 x g for 15 sec. Discard the flow-through.
9. Add 500 µl RNA wash buffer and spin at 9,600 x g for 2 min. Discard the flow-through.
10. Place the spin column in a new 2 ml collection tube and centrifuge at 9,600 x g for 1 min. Discard the flow-through.
11. Add 30-50 µl RNase-free water directly to the spin column and centrifuge at 9,600 x g for 1 min. Store RNA at -80 °C.
12. Perform RNAseq analysis¹⁴.