NOTE: All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee.

1. Animal Preparation

1. Prepare 6 C57BL/6 control mice for the DFCO measurement, by anesthetizing them with ketamine and xylazine as outlined in step 2.3 below.
2. Prepare all of the other mice with the different lung pathologies shown in Table 1 by using the same procedure as for the controls. Specific details needed to establish each of these models are found in the relevant references. Control mice and those in the other pathologic cohorts are all 6-12 weeks of age.

2. Measurement of Diffusion Factor for Carbon Monoxide (DFCO)

1. Set up the gas chromatograph module supplied with the machine to measure peaks for nitrogen, oxygen, neon, and carbon monoxide. For this application use only the neon and CO data.
   NOTE: This instrument uses a molecular sieve column with helium as carrier gas, with a 12.00 µm film, 320.00 µm ID and 10 m length. The chromatograph column has a volume of 0.8 ml, but we used 2 ml to ensure adequate clearing of the connecting tubing with the sample.
2. At the start of each experimental day, prior to making measurements of the samples from the mice, take a 2 ml sample directly from a gas mixture bag containing approximately 0.5% Ne, 0.5% CO, and balance air, and use this sample to calibrate the gas chromatograph.
3. Anesthetize mice with ketamine (90 mg/kg) and xylazine (15 mg/kg), and confirm anesthesia by the absence of reflex motion. Apply veterinary ointment on the eyes to prevent dryness. Tracheostomize the mice with a stub needle cannula (18 G in adults or 20 G in very young mice).
   NOTE: The DFCO is completed in less than 10 min after anesthesia and prior to any mechanical ventilation or other procedures.
4. In mice greater than 6 weeks of age, use a 3 ml syringe to withdraw 0.8 ml of gas from the gas mixture bag. Connect the syringe to the tracheal cannula and quickly inflate the lung. Using a metronome, count 9 sec, and then quickly withdraw the 0.8 ml (exhaled air).
5. Dilute this withdrawn 0.8 ml exhaled air to 2 ml with room air, allow it to rest for at least 15 sec. Then inject the whole sample into the gas chromatograph for analysis.
6. While analyzing this first DFCO sample, inflate the mouse lung with a second 0.8 ml from the gas mixture bag, and then process this sample identical to the first sample. Average the two DFCO measurements.
   NOTE: For measurements in mice as young as 2 weeks of age, use a volume of 0.4 ml, since 0.8 ml is too large a volume to make measurements in lungs of very young mice. It is better to use the 0.8 ml volume for mice older than 6 weeks, and that if the 0.4 ml volume is needed for some mice, it should be used consistently for all mice in the cohort being studied.
7. Calculate DFCO as 1 – (CO₉ / CO_c) / (Ne₉ / Ne_c), where c and 9 subscripts refer to concentrations of the calibration gases injected and the gases removed after a 9 sec breath hold time, respectively.
8. Analyze and compare differences with a one-way ANOVA and assess the significance level with Tukey’s correction for multiple comparisons in all the cohort mice. Consider p <0.05 as significant value.
   NOTE: All of the mice used here were part of experimental studies involving several subsequent measurements of lung ventilation, mechanics, lung lavage, or histology, which are not reported here. In addition, since the method is the same in all the experimental models as was done above in the control mice, only the results from the various pathologic models are presented. Further information on these models is presented in the supplemental table.
9. Euthanize the animals by deep anesthetic overdose followed by cervical dislocation or decapitation. Where needed, remove the lung cells and/or tissues from the dead mice for further biologic or histologic processing and analysis.