Aerosol inhalation exposure system

Male and female age-matched mice aged 8−10 weeks were infected with Mtb H37Rv either by intratracheal instillation or by an aerosol inhalation exposure system (Glas-Col, USA). Lung CFU at 24 h post infection was used to determine initial dose of infection. For time course infection studies, mice were sacrificed at 3, 8 and 14 weeks post infection. The number of viable bacteria present in lungs and spleens was enumerated by plating serial dilutions of homogenates on BBL 7H11 agar plates supplemented with carbencillin (50 µg/ml) and cycloheximide (50 µg/ml). CFU were enumerated as described above. For survival studies, 10 mice/group were used, and time-to-death was observed and represented by a Kaplan−Meier graph. Mice were monitored for survival until 300 days whereas all Cbs^+/− mice died by day 270. No deaths were observed in uninfected WT or Cbs^+/− mice within 1 year of the end of the survival experiment.

Animals were divided into three different groups: healthy controls (Mtb⁻, n = 6), infected mice four weeks post-infection (Mtb⁺4w, n = 6) and infected mice twelve weeks post-infection (Mtb⁺12w, n = 5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All procedures were conducted according to NIH guidelines and protocols were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120–250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 h post-infection. Mice were sacrificed using anesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1× PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1× PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N₂(l)-containing recipient to freeze the tissues and stored at −80 °C to avoid postmortem metabolic processes.